Methods for identifying compounds which modulate circadian rhythm

ABSTRACT

The invention is based, in part, on the discovery that the CRY proteins and the PER2 protein function as important modulators of mammalian circadian rhythm. The invention includes methods of modulating the circadian rhythm and identifying compounds that modulate the circadian rhythm.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from U.S. ProvisionalApplication Serial No. 60/203,005 filed May 10, 2000, and U.S.Provisional Application Serial No. 60/145,363, filed Jul. 22, 1999,these applications are incorporated herein by reference in theirentirety.

FIELD OF THE INVENTION

[0002] The field of the invention relates to the regulation of circadianrhythms.

BACKGROUND OF THE INVENTION

[0003] Circadian rhythms in mammals are regulated by a master clocklocated in the suprachiasmatic nucleus (SCN) of the brain (Klein et al.,Suprachiasmatic nucleus: The Mind's Clock, Oxford University Press, NewYork, 1991; Reppert and Weaver, Cell 89:487-490, 1997). Environmentallight-dark cycles entrain the SCN clock to the 24-hr day via direct andindirect retinal projections. The timekeeping capability of the SCN isexpressed at the level of single neurons (Welsh et al., Neuron14:697-706, 1995).

[0004] The SCN clock mechanism is cell-autonomous, possibly based ontranscriptional and translational negative feedback loops (Reppert,Neuron 21:1-4, 1998). Precedent for such a mechanism has been describedfor circadian clocks in the fly Drosophila melanogaster.

[0005] In the fly, autoregulatory transcriptional loops occur in whichprotein products of clock genes periodically enter the nucleus tosuppress their own transcription. This feedback loop involves dynamicregulation of the clock genes period (per) and timeless (Tim). As thelevels of PER and TIM rise, they are phosphorylated, form heterodimers,and are then translocated to the nucleus where they negatively regulatetheir own transcription (Saez and Young, Neuron 17:1-920, 1996;Darlington et al., Science 280:1599-1603, 1998). Negativetranscriptional regulation appears to involve interference withdrosophila CLOCK:drosophila dBMAL-1 (dCLOCK:dBMAL-1) and may be mediatedby direct interaction of PER and TIM with dCLOCK. dCLOCK and dBMAL-1 arepositive factors which drive Per and Tim transcriptional activation bybinding to CACGTG E-box enhancers in the promoters of Per and Tim(Allada et al., Cell 93:791-804, 1998; Rutila et al., Cell 93:805-814,1998; Darlington et al., supra; Hao et al., Mol. Cell Biol.17:3687-3693, 1997). The temporal phosphorylation of PER provides atleast part of the time delay between transcription and PER-TIM negativefeedback necessary to sustain a 24-hr molecular oscillation indrosophila (Price et al., Cell 94:83-95, 1998).

SUMMARY OF THE INVENTION

[0006] The invention is based, in part, on the discovery that the coreclockwork in the SCN is comprised of interacting feedback loops. It wasdiscovered that cryptochrome (CRY) proteins are critical players in thenegative limb of the mammalian clock feedback loop and Period 2 (PER2)protein is a critical regulator of the Bmal-1 loop. The CRY proteins andPER2 protein therefore function as important modulators of mammaliancircadian rhythm.

[0007] It was discovered that mammalian CRY proteins can translocatefrom the cytoplasm to the nucleus of a cell and inhibit CLOCK:BMAL-1induced transcription. It was also discovered that CRY proteins canhomodimerize or heterodimerize with other circadian proteins. Theability of CRY to heterodimerize with other proteins provides amechanism whereby CRY can modulate the activity of other circadianproteins. For example, mouse CRY proteins can function as dimeric andpotentially trimeric partners for mouse PER proteins; these interactionslead to the nuclear translocation of PER. Once in the nucleus, PER caninhibit CLOCK:BMAL-1 induced transcription. In addition, it wasdiscovered that mouse CRY can form heterodimeric complexes with mouseTIM. The interaction of TIM with CRY may have a role in modulating thenegative feedback of mouse PER and/or mouse CRY rhythms. Thus, thecompounds which can disrupt the interaction of CRY with itself and othercircadian proteins can be used to reset the circadian clock.

[0008] In addition, it was discovered that PER2 positively regulatestranscription of the Bmal-1 gene. The ability of PER2 to positivelyregulate the transcription of Bmal-1 indicates that PER2 controls therhythmic regulation of Bmal-1. The availability of BMAL-1 is criticalfor restarting the circadian clock loop. When BMAL-1 is available, itheterodimerizes with CLOCK, thereby driving the transcription of Pergenes (e.g., in the mouse(m), mPER1-3) and Cryptochrome genes (e.g.,mouse mCry1 and mCry2). Compounds which can disrupt the ability of PER2to positively activate Bmal-1, or compounds which can modulatetranscription of Bmal-1, can be used to reset the circadian clock.

[0009] Accordingly, the invention includes a method for identifying acompound which binds to a mammalian CRY protein. The method, which isuseful as a quick initial screen for CRY agonists and antagonists,includes contacting the CRY protein with a test compound and determiningwhether the latter binds to the CRY protein. Binding by the testcompound to the CRY protein indicates that the test compound is a CRYprotein binding compound. For ease of detection, the test compound canbe labeled, e.g., radiolabeled. The CRY protein can any mammalian CRYprotein such as a CRY from a mouse, rat, rabbit, goat, horse, cow, pig,dog, cat, sheep, pig, non-human, primate, or human. In particular, theCRY protein is a mouse CRY1 or CRY2 or human CRY1 or CRY2.

[0010] The method may further include contacting the test compound with:a CRY protein in the presence of a PER protein; a CRY protein in thepresence of a TIM protein; a CRY protein in the presence of aCLOCK:BMAL-1 complex; or a CRY protein in the presence of a BMAL-1protein; and determining whether the test compound disrupts theassociation of the CRY protein with the PER, TIM, CLOCK:BMAL-1, orBMAL-1 protein, as the case may be; wherein a decrease in theassociation in the presence of the test compound compared to theassociation in the absence of the test compound indicates that the testcompound disrupts the association of the CRY protein with the indicatedbinding partner. The PER protein can any mammalian PER protein such asmouse, rat, rabbit, goat, horse, cow, pig, dog, cat or human. Forexample, the PER protein may be mouse or human PER1, PER2 or PER3.

[0011] The method can further include contacting the test compound withthe first CRY protein in the presence of a second CRY protein anddetermining whether the test compound disrupts the association of thefirst CRY protein with the second CRY protein, wherein the second CRYprotein has an amino acid sequence the same as or different than thefirst CRY protein, and wherein a decrease in the association in thepresence of the test compound compared to the association in the absenceof the test compound indicates that the test compound disrupts theassociation of the first CRY protein and the second CRY protein. Thefirst and second CRY proteins can be any mammalian CRY protein such as aCRY from a mouse, rat, rabbit, goat, horse, cow, pig, dog, cat, sheep,non-human, primate or human. For example, each CRY protein can be amouse or human CRY1 or CRY2 and the second CRY protein is a mouse CRY1or CRY2.

[0012] The method can further include providing a cell or cell-freesystem which includes a CRY protein, a CLOCK:BMAL-1 complex, and a DNAcomprising an E-box operatively linked to a reporter gene. The methodincludes introducing the test compound into the cell or cell-free systemand assaying for transcription of the reporter gene, wherein an increasein transcription in the presence of the compound compared totranscription in the absence of the compound indicates that the compoundblocks CRY-induced inhibition of CLOCK:BMAL-1-mediated transcription ina cell. The cell can be any cell type, such as a cultured mammaliancell, e.g., a NIH3T3 cell, a COS7 cell, or a clock neuron. The reportergene can be a gene that encodes a detectable marker, e.g., luciferase.

[0013] The invention further includes a method for identifying acompound which disrupts the association of a CRY protein and a secondprotein or protein complex, which can be any of the following: a PERprotein, a TIM protein, a BMAL-1 protein, a second CRY protein, or aCLOCK:BMAL-1 complex. The method includes contacting a test compoundwith the CRY protein in the presence of the second protein (or proteincomplex) and determining whether the test compound disrupts theassociation of the CRY protein and the second protein (or proteincomplex), wherein a decrease in the association in the presence of thetest compound compared to the association in the absence of the testcompound indicates that the test compound disrupts the association ofthe CRY protein and the second protein. The first and second CRYproteins can be any mammalian CRY protein such as a CRY protein from amouse, rat, rabbit, goat, horse, cow, sheep, pig, dog, cat, non-humanprimate or human, e.g., a mouse or human CRY1 or CRY2. The PER proteincan be any mammalian PER protein as described above, e.g., a mouse PER1,PER2 or PER3. The TIM protein can be any mammalian TIM protein asdescribed above, e.g., a mouse or human TIM protein. The CLOCK and theBMAL-1 proteins can be any mammalian CLOCK and BMAL-1 proteins asdescribed above, particularly mouse or human.

[0014] Also within the invention is a method for identifying a compoundthat blocks CRY-induced inhibition of CLOCK:BMAL-1 transcription in acell. The method includes providing a cell comprising a CRY protein, aCLOCK:BMAL-1 complex, and a DNA comprising an E-box operatively linkedto a reporter gene; introducing the compound into the cell or acell-free transcription system; and assaying for transcription of thereporter gene, wherein an increase in transcription in the presence ofthe compound compared to transcription in the absence of the compoundindicates that the compound blocks CRY-induced inhibition ofCLOCK:BMAL-1-mediated transcription. The cell can be any cell type, suchas a cultured mammalian cell, e.g., a NIH3T3 cell, a COS7 cell or aclock neuron. The reporter gene can be gene that encodes a detectablemarker, e.g., luciferase.

[0015] The invention further includes a method for identifying acompound that activates or inhibits the transcription of Per2. Themethod includes providing a cell including a mammalian Per2 regulatorysequence operatively linked to a reporter gene, introducing a testcompound into the cell, and assaying for transcription of the reportergene in the cell. A decrease in transcription in the presence of thecompound compared to transcription in the absence of the compoundindicates that the compound inhibits Per2 transcription in a cell.Likewise, an increase of transcription in the presence of the compoundcompared to transcription in the absence of the compound indicates thatthe compound inhibits Per2 transcription in a cell. The cell can be anycell that can generate circadian rhythms, such as a NIH3T3 cell, a Cos-7cell or a clock neuron. The reporter gene can be any detectable marker,e.g., a luciferase, a chloramphenicol acetyl transferase, abetagalactosidase, an alkaline phosphate, or a fluorescent protein suchas green fluorescent protein. The Per2 regulatory sequence can be anymammalian Per2 regulatory sequence, e.g., from a mouse, a rat, a rabbit,a goat, a horse, a cow, a pig, a dog, a cat, a sheep, a non-humanprimate, or a human. In particular, the Per2 regulatory sequence can bea mouse Per2 regulatory sequence (SEQ ID NO:3).

[0016] Also within the invention is a method of determining if acandidate compound positively regulates the expression of Bmal-1. Themethod includes providing a transgenic animal whose somatic and germcells comprise a disrupted Per2 gene, the disruption being sufficient toinhibit the ability of Per2 to positively regulate Bmal-1, administeringa test compound to the mouse, and detecting Bmal-1 expression, whereinan increase in the expression of Bmal-1 indicates that the compound canpositively regulate expression of Bmal-1.

[0017] The invention also features a method of modulatingcircadian-clock controlled rhythms in a cell including comprisingintroducing into a cell an expression vector encoding a BMAL-1 proteinsuch that an effective amount of the BMAL-1 protein is produced in thecell, thereby modulating circadian-clock controlled rhythms. The BMAL-1can be any mammalian BMAL-1, e.g., that of a mouse, a rat, a rabbit, agoat, a horse, a cow, a dog, a cat, a sheep, a non-human primate, or ahuman BMAL-1.

[0018] Also within the invention is a method of modulatingcircadian-clock controlled rhythms in a cell comprising introducing intothe cell an effective amount of an oligonucleotide antisense to a part,or all, of a mammalian Bmal-1, thereby inhibiting expression of Bmal-1in the cell and modulating circadian-clock rhythms. Oligonucleotides canbe antisense to any mammalian Bmal-1, e.g., Bmal-1 from a mouse, a rat,a rabbit, a goat, a horse, a cow, a sheep, a non-human primate, or ahuman.

[0019] The invention further includes isolated nucleic acid moleculeswhich are at least about 60% (or 65%, 75%, 85%, 95%, or 98%) identicalto the nucleotide sequence of mouse TIMELESS (TIM) (SEQ ID NO:1). Theinvention also features isolated nucleic acid molecules which include afragment of at least 100 (e.g., at least 200, 300, 400, 500, 600, 700,800, 900, 1000, 1500, 2000, 2500, 3000, 3500, or 3745) nucleotides ofthe nucleotide sequence of SEQ ID NO:1, or a complement thereof. Theinvention also features nucleic acid molecules which include anucleotide sequence encoding a protein having an amino acid sequencethat is at least about 60% (or 70%, 75%, 85%, 95%, or 98%) identical tothe amino acid sequence of SEQ ID NO:2. In a preferred embodiment, theisolated nucleic acid molecule has the nucleotide sequence of SEQ ID NO:1, or a complement thereof.

[0020] Also within the invention is an isolated polypeptide having anamino acid sequence that is at least about 60%, preferably 70%, 75%,85%, 95%, or 98%, identical to the amino acid sequence of SEQ ID NO:2.Also within the invention are isolated polypeptides encoded by a nucleicacid molecule having a nucleotide sequence which hybridizes understringent hybridization conditions to the complement of SEQ ID NO: 1.

[0021] The invention also features isolated nucleic acid molecules whichare at least about 60% (or 65%, 75%, 85%, 95%, or 98%) identical to themouse Per2 upstream sequence (SEQ ID NO:3) containing a sequencecontrolling expression of mouse Per2. The invention also featuresisolated nucleic acid molecules which include a fragment of at least 100(e.g., at least 200, 300, 400, 500, 600, 700, 800, 900, or 950)nucleotides of the nucleotide sequence of SEQ ID NO:3, or a complementthereof.

[0022] The invention also includes nucleic acid molecules that hybridizeunder stringent conditions to a nucleic acid molecule having thenucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3. The nucleic acidmolecules can be, for example, at least 20 (e.g. at least about 30, 40,50, 70, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500,2000, 2500, 3000, 3500, or 3745) nucleotides in length.

[0023] Another aspect of the invention provides vectors, e.g.,recombinant expression vectors, comprising a nucleic acid moleculedescribed herein. The vector or nucleic acid molecule can be provided ina host cell. Such cells may be utilized for producing a polypeptide ofthe invention by culturing the cells in a suitable medium.

[0024] Also within the invention are a substantially pure preparation ofa mouse or human TIM, a mouse or human CRY:PER heterodimer, a CRY:TIMheterodimer, and a mammalian CRY:CRY homodimer.

[0025] Isolated antibodies, which specifically bind to mouse CRY, mousePER, mouse TIM, mouse BMAL-1 are also within the invention.

[0026] As used herein, “isolated DNA” means either DNA with anon-naturally occurring sequence or DNA free of the genes that flank theDNA in the genome of the organism in which the DNA naturally occurs. Theterm therefore includes a recombinant DNA incorporated into a vector,into an autonomously replicating plasmid or virus, or into the genomicDNA of a prokaryote or eukaryote. It also includes a separate moleculesuch as a cDNA, a genomic fragment, a fragment produced by polymerasechain reaction (PCR), or a restriction fragment.

[0027] As used herein, an regulatory sequence which is “operably linked”to a second sequence (or vise versa) means that both are incorporatedinto a genetic construct so that the regulatory sequence effectivelycontrols expression of a second sequence.

[0028] As used herein, a “substantially pure” protein refers to aprotein which either (Klein et al., (1991). Suprachiasmatic nucleus: TheMind's Clock, Oxford University Press, New York. has a non-naturallyoccurring sequence (e.g., mutated, truncated, chimeric, or completelyartificial), or (D. R. Weaver, J. Biol. Rhythms 13, 100 (1998) has anaturally occurring sequence but is not accompanied by or at leastpartially separated from, components that naturally accompany it.Typically, the protein is substantially pure when it is at least 60% (byweight) free from the proteins and other naturally-occurring organicmolecules with which it is naturally associated. Preferably, the purityof the preparation is at least 75%, more preferably at least 90%, andmost preferably at least 99%, by weight. A substantially pure proteincan be obtained, for example, by extraction from a natural source, byexpression of a recombinant nucleic acid encoding the protein or bychemical synthesis. Purity can be measured by any appropriate method,e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLCanalysis. A chemically synthesized protein or a recombinant proteinproduced in a cell type other than the cell type in which it naturallyoccurs is, by definition, substantially free from components thatnaturally accompany it. Accordingly, substantially pure proteins includethose having sequences derived from eukaryotic organisms but synthesizedin E. coli or other prokaryotes.

[0029] As used herein, the term “vector” refers to a replicable nucleicacid construct. Examples of vectors include plasmids and viral nucleicacids.

[0030] As used herein, a “circadian protein” refers to a protein thatparticipates in the circadian timing system and controls circadianrhythm. Examples of circadian proteins include PER, TIM, CLOCK, andBMAL-1.

[0031] As used herein, an antibody that “specifically binds” a mouse orhuman CRY, PER or TIM, respectively, is an antibody that binds only tomouse or human CRY, PER or TIM and does not bind to (i) other moleculesin a biological sample or (ii) CRY, PER or TIM of another organism.

[0032] As used herein, a “therapeutically effective amount” is an amountof the nucleic acid of the invention which is capable of producing amedically desirable result in a treated animal.

[0033] As used herein, a “reporter gene” means a gene whose expressioncan be assayed.

[0034] As used herein, the terms “heterologous DNA” or “heterologousnucleic acid” is meant to include DNA that does not occur naturally aspart of the genome in which it is present, or DNA which is found in alocation or locations in the genome that differs from that in which itoccurs in nature, or occurs extra-chromasomally, e.g., as part of aplasmid.

[0035] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention pertains. The preferred methodsand materials are described below, although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention. All publications, patentapplications, patents, and other references mentioned herein areincorporated by reference in their entirety. In case of conflict, thepresent document, including definitions, will control. Unless otherwiseindicated, materials, methods, and examples described herein areillustrative only and not intended to be limiting.

[0036] Various features and advantages of the invention will be apparentfrom the following detailed description and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0037]FIG. 1 is a histogram showing dose-response studies on inhibitionof CLOCK:BMAL-1-induced transcription by the mPER and mTIM proteins.

[0038] FIGS. 2A-2B are line graphs showing mouse Cry1 and Cry2 mRNAlevels in SCN (FIG. 2A) and mouse Cry1 and Cry2 RNA levels in skeletalmuscle (FIG. 2B).

[0039] FIGS. 3A-D is a histogram showing inhibition ofCLOCK:BMAL1-mediated transcription from the vasopressin (AVP) promoter(FIGS. 3A, 3C-D) or mPer1 promoter (FIG. 3B) by mPER1, mCRY1 and mCRY2(250 ng each).

[0040]FIG. 4 is a schematic representation of epitope-tagged mouse CRY1and CRY2 proteins evaluated for cellular location and inhibition ofClock:Bmal-1 mediated transcription.

[0041] FIGS. 5A-D are histograms depicting the specificity of mouse PERand mouse CRY in inhibiting transcription of Mop4:Bmal-1 mediatedtranscription.

[0042]FIG. 6 is a representation of the nucleotide sequence of mouse TIM(SEQ. ID NO:1).

[0043]FIG. 7 is a representation of the amino acid sequence of mouse TIM(SEQ. ID NO:2).

[0044]FIG. 8 is a representation of the nucleotide sequence of theregulatory sequence of mouse Per2 (SEQ. ID NO:3).

[0045]FIG. 9 is a line graph depicting temporal profiles of Bmal1 RNAlevels in the SCN of wild type (solid) and Clock/Clock (dashed) mice.Each value is the mean±SEM of 5-9 animals. Data at CT 2, 3, 22, and 24are double-plotted. Gray bar, subjective day; black bar, subjectivenight.

[0046]FIG. 10 is a line graph depicting CLOCK mRNA levels in the SCN ofwild-type (solid line) or Clock/Clock (dashed line) mice. Each value isthe mean±SEM of 5-9 animals. Data at CT 2, 3, 22, and 24 aredouble-plotted. Gray bar, subjective day; black bar, subjective night.

[0047]FIG. 11 is a line graph depicting temporal profiles of Bmal1 RNAlevels in the SCN of wild-type (solid line) and mPER2^(Brdm1) mutant(dashed line) mice. Each value is the mean±SEM of 4 animals.

[0048]FIG. 12 is a line graph depicting temporal profiles of mCry1 RNAlevels in the SCN of wildtype (solid line) and mPER2^(Brdm1) mutant(dashed line) mice are shown. Each value is the mean±SEM of 4 animals.

[0049]FIG. 13 is a schematic representation of different mPER2constructs with a V5 epitope tagged at the carboxyl terminus of mPER2.Also shown is the cellular location of immunofluorescence of V5-taggedmPER2 constructs expressed in COS-7 cells either with (+) or without (−)mCRY1. The cellular location of immunofluorscence was scored as one ofthree categories: cytoplasm only (C), both cytoplasm and nucleus (B), ornucleus only (N). Values shown are the mean percentages from twoexperiments; all values were within 17% of the mean. Gray bars are PASdomain.

[0050]FIG. 14 is a histogram depicting attenuated peak levels of Bmal1RNA in mCry-deficient mice. Quantitation of Bmal1 RNA levels in the SCNof wild-type (solid bars) and mCry-deficient (open bars) mice. Valuesare the mean±SEM of 5 animals. Mice were studied on the first day inDD. * is the significance difference in Bmal1 RNA levels between CT 6and CT 18 in wild-type mice; P<0.0001.

[0051]FIG. 15 is a histogram depicting quantitation of Clock RNA levelsin the SCN of wildtype (solid bars) and mCry-deficient (open bars) mice.Values are the mean±SEM of 5 animals.

[0052]FIG. 16 is a histogram depicting the effects of mCRY proteins ontranscriptional activation in Drosophila S2 cells. Values are luciferaseactivity expressed as relative to the response in presence of activators(100%). Each value is the mean±SEM of three replicates from a singleassay.

[0053]FIG. 17 is a histogram depicting the effects of mCRY proteins ontranscriptional activation in COS-7 cells. Presence (+) or absence (−)of luciferase reporter (pGL3-Basic) (10 ng) and expression plasmids(0.25 ug mClock, shBmal1, hMop4, dclock; 0.1 ug mCry1, mCry2) isdenoted. Values are luciferase activity expressed as relative to theresponse in presence of activators (100%). Each value is the mean±SEM ofthree replicates from a single assay. The results shown arerepresentative of three independent experiments.

[0054]FIG. 18 is a schematic drawing depicting a model of circadianclockwork within an individual SCN neuron.

DETAILED DESCRIPTION

[0055] It has been discovered that members of the mouse PER family(PER1, PER2, and PER3), the mouse CRY family (CRY1, and CRY2) and mouseTIM can interact directly with each other. The ability of these proteinsto interact is critically involved in regulating circadian rhythm. Morespecifically, PER, CRY and TIM control circadian rhythm by inhibitingthe transcriptional feedback loop which is at the heart of the mammaliancircadian clock.

[0056] It was also discovered that PER2 positively regulates thetranscription of Bmal-1, thereby controlling the rhythmic regulation ofBmal-1. BMAL1-1 functions as a positive regulator in the circadian loop.More specifically, BMAL-1 forms a heterodimeric protein with CLOCK,which heterodimer in turn positively regulates the expression of thecircadian genes such as PER or CRY.

[0057] Based on the discovery made herein, the SCN clockwork ispredicted to include three types of interacting molecular loops (FIG.18). The Cry genes comprise one loop that has true autoregulatory,negative feedback features, with the protein products feeding back toturn off their transcription. The second loop is that manifested by eachof the Per genes and some clock-controlled genes (CCGs) (for example,vasopressin prepropressophysin). This loop type is driven by the samepositive elements (CLOCK(C):BMAL1(B)) as the CRY loop, but is not turnedoff by the respective gene products. Instead, these loops use the CRYproteins as negative regulators, leaving the generated protein productsfree to transduce other actions. For example, PER2 is used for thepositive transcriptional regulation of the Bmal-1 gene. The rhythmicregulation of Bmal-1 comprises the third loop, whose rhythmicity iscontrolled by the cycling presence and absence of a positive elementdependent upon mPER2. This positive feedback loop functions to augmentthe positive regulation of the first two loops.

[0058] This model of interacting loops proposes that at the start of thecircadian day PER and CRY transcription are driven by accumulatingCLOCK:BMAL1 heterodimers acting through E box enhancers. After a delay,the PER and CRY proteins are synchronously expressed in the nucleuswhere the CRY proteins shut off Clock:Bmal1-mediated transcription bydirectly interacting with these transcription factors. At the same timethat the CRY proteins are inhibiting Clock:Bmal-1-mediatedtranscription, PER2 either shuttles a transcriptional activator into thenucleus or coactivates a transcriptional complex to enhance Bmal-1transcription. The importance of the Bmal-1 RNA rhythm is to drive aBmal-1 rhythm after a 4 to 6 hour delay. This delay in the proteinrhythm would provide increasingly available CLOCK:BMAL1 heterodimers atthe appropriate circadian time to drive Per and Cry transcription,thereby restarting the cycle. It is thus predicted that BMAL-1availability is rate limiting for heterodimer formation and critical forrestarting the loops.

[0059] TIM Nucleic Acid Molecules

[0060] The invention pertains to isolated nucleic acid molecules thatencode mouse TIM proteins or biologically active portions thereof, aswell as nucleic acid molecules which can serve as hybridization probesto identify TIM-encoding nucleic acids (e.g., TIM mRNA), or as PCRprimers for the amplification or mutation of TIM nucleic acid molecules.The nucleic acid encoding mouse TIM (SEQ ID NO:1) (and/or the complementof that nucleic acid) can be used as a probe to identify nucleic acidsrelated to the mouse TIM gene, e.g., other naturally occurring mammalianTIM DNA's.

[0061] Fragments of SEQ ID NO:1 and its complement can be used as probesor primers, so long as they are at least 10, and preferably at least 15(e.g., at least 18, 20, 25, 50, 100, 150, or 200) nucleotides in length.TIM probes and primers can be produced using any of several standardmethods (see, e.g., Ausubel et al., 1989, Current Protocols in MolecularBiology, Vol. 1, Green Publishing Associates, Inc., and John Wiley &Sons, Inc., NY). For example, the probe can be generated using PCRamplification methods in which oligonucleotide primers are used toamplify a portion of SEQ ID NO:1 that can be used as a specific probe.Such probes and primers are part of the invention.

[0062] Hybridization under stringent conditions can be used to identifynucleic acid sequences which encode mouse TIM or other related TIMs,e.g., other mammalian TIM proteins. A related nucleic acid sequence hasat least 50% sequence identity to mouse TIM cDNA (SEQ ID NO:1). Standardhybridization conditions (e.g., moderate or highly stringent conditions)are known to those skilled in the art and can be found in CurrentProtocols in Molecular Biology, John Wiley & Sons, N.Y. (1989),6.3.1-6.3.6, hereby incorporated by reference. Moderate hybridizationconditions are defined as equivalent to hybridization in 2×sodiumchloride/sodium citrate (SSC) at 30° C., followed by one or more washesin 1×SSC, 0.1% SDS at 60° C. Highly stringent conditions are defined asequivalent to hybridization in 6× sodium chloride/sodium citrate (SSC)at 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65° C.

[0063] Nucleic acids which hybridize to the above-described probes understringent conditions can be used as probes themselves to analyze theexpression of mouse TIM mRNA in the SCN. These nucleic acids can also beused to express mouse TIM polypeptides or immunogenic fragments thereoffor raising mouse TIM antibodies.

[0064] Genomic fragments of the TIM locus that are hybridizable to theabove-described probes are also included in the invention. Suchfragments are useful starting materials for generating, e.g., knockoutconstructs that are used to create non-human transgenic mammalscontaining null mutations at the TIM locus.

[0065] The invention further encompasses nucleic acid molecules thatdiffer from the nucleotide sequence of SEQ ID NO:1 due to degeneracy ofthe genetic code, and thus encode the same TIM protein as that encodedby the nucleotide sequence shown in SEQ ID NO:1.

[0066] Mutations which change the nucleotide sequence of SEQ ID NO:1without altering the functional activity of the TIM protein are alsowithin the scope of the invention. For example, one can make nucleotidesubstitutions leading to amino acid substitutions at “non-essential”amino acid residues. A “non-essential” amino acid residue is a residuethat can be altered from the wild-type sequence of mouse TIM (e.g., thesequence of SEQ ID NO:2) without altering the biological activity,whereas an “essential” amino acid residue is required for biologicalactivity. For example, amino acid residues that are conserved among theTIM proteins of various species are predicted to be particularlyunamenable to alteration. These can be identified by sequence comparisonamong the known TIM proteins (yeast, Drosphylia and now, mouse) Thus,the invention encompasses nucleic acid molecules encoding mouse TIMproteins that contain changes in amino acid residues that are notessential for activity. Such TIM proteins differ in amino acid sequencefrom SEQ ID NO:2, yet retain biological activity.

[0067] An isolated nucleic acid molecule encoding a TIM protein having asequence which differs from that of SEQ ID NO:2 can be created byintroducing one or more nucleotide substitutions, additions or deletionsinto the nucleotide sequence of SEQ ID NO:1 such that one or more aminoacid substitutions, additions or deletions are introduced into theencoded protein. Mutations can be introduced by standard techniques,such as site-directed mutagenesis and PCR-mediated mutagenesis.Generally, additions or deletions of nucleotides will be done inmultiples of three, so as to avoid a frame shift.

[0068] TIM Polypeptides

[0069] A mouse TIM polypeptide can be isolated and purified from anatural source. Alternatively, it can be produced recombinantly orchemically synthesized by conventional methods. A TIM polypeptide,full-length or truncated, can also be part of a fusion protein, forexample, by linking it to an antigenic determinant to facilitatepurification. The TIM polypeptides can be prepared for a variety ofuses, e.g., generation of antibodies which can be used to detect TIM,and in screening assays which identify compounds that disrupt theassociation of TIM with CRY.

[0070] Techniques for generating substantially pure polypeptidepreparations are well known in the art. A typical method involvestransfecting host cells (e.g., bacterial cells such as E. coli, ormammalian cells such as COS7) with an expression vector carrying anucleic acid that encodes a mouse TIM protein. The recombinantpolypeptide so produced can be purified from the culture medium or fromlysates of the cells.

[0071] Conventional site-directed mutagenesis techniques can be appliedto a TIM coding sequence, e.g., SEQ ID NO:1, to generate TIM sequencevariants optimized for expression in a given type of host cell.

[0072] Furthermore, one skilled in the art can prepare not only anatural mouse TIM protein with a naturally occurring sequence (SEQ IDNO:2), but also proteins with substantially the same function as that ofthe natural protein, by replacing amino acids in the protein. Methodsfor amino acid alteration include, for example, a site-directedmutagenesis system using PCR (GIBCO-BRL, Gaithersburg, Md.); theoligonucleotide-mediated site-directed mutagenesis method (Kramer,Methods in Enzymol. 154:350-367 1997); and the Kunkel method (MethodsEnzymol. 85:2763-2766, 1988). Usually ten or fewer, preferably six orfewer, and more preferably three or fewer amino acids (e.g., one or two)are substituted. Proteins functionally equivalent to the TIM protein canbe produced by conservative amino acid substitutions at one or moreamino acid residues. A “conservative amino acid substitution” is one inwhich the amino acid residue is replaced with an amino acid residuehaving a chemically similar side chain. Families of amino acid residueshaving similar side chains have been defined in the art. These familiesinclude amino acids with basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., glycine, asparagine, glutamine,serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g.,alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine).

[0073] Biologically active portions of a mouse TIM protein includepeptides comprising amino acid sequences identical to or derived fromthe amino acid sequence of the mouse TIM protein (e.g., the amino acidsequence shown in SEQ ID NO:2).

[0074] A TIM protein that has a high sequence identity to SEQ ID NO:2 isalso included in the invention. A useful TIM protein has an amino acidsequence at least 60% identical, preferably at least 70%, morepreferably at least 80%, and even more preferably at least 90, 95, 96,97, 98 or 99% identical to the amino acid sequence of SEQ ID NO:2, andretains the functional activity of the TIM protein of SEQ ID NO:2.

[0075] To determine the percent sequence identity of two amino acidsequences or of two nucleic acids, the sequences are aligned for optimalcomparison purposes (e.g., gaps can be introduced in the sequence of afirst amino acid or nucleic acid sequence for optimal alignment with asecond amino or nucleic acid sequence). The amino acid residues ornucleotides at corresponding amino acid positions or nucleotidepositions are then compared. When a position in the first sequence isoccupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent homology between the twosequences is a function of the number of identical positions shared bythe sequences (i.e., % identity=# of identical positions/total # ofpositions (e.g., overlapping positions)×100). In one embodiment, the twosequences are the same length.

[0076] To determine percent homology between two sequences, thealgorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA87:2264-2268, 1990), modified as in Karlin and Altschul (Proc. Natl.Acad. Sci. USA 90:5873-5877, 1993), is used. Such an algorithm isincorporated into the NBLAST and XBLAST programs of Altschul et al. (J.Mol. Biol. 215:403-410, 1990. BLAST nucleotide searches are performedwith the NBLAST program, score=100, wordlength=12 to obtain nucleotidesequences homologous to a nucleic acid molecules of the invention. BLASTprotein searches are performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to mouse TIMprotein. To obtain gapped alignments for comparison purposes, GappedBLAST is utilized as described in Altschul et al. (Nucleic Acids Res.25:3389-3402, 1997). When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and NBLAST)are used. See http://www.ncbi.nlm.nih.gov.

[0077] Per2 Regulatory Sequence

[0078] The invention pertains to an isolated genomic nucleic acidmolecule that includes the mouse Per2 regulatory sequence(promoter/enhancer sequence), as well as nucleic acid molecules whichcan serve as hybridization probes to identify a Per2 regulatorysequence, or as PCR primers for the amplification or mutation of a Per2regulatory sequence.

[0079] Fragments of mouse Per2 regulatory sequence (SEQ ID NO:3) and itscomplement can be used as probes or primers, so long as they are atleast 10, and preferably at least 15 (e.g., at least 18, 20, 25, 50,100, 150, or 200) nucleotides in length. PER2 regulatory sequence probesand primers can be produced using any of several standard methodsdescribed above. For example, the probe can be generated using PCRamplification methods in which oligonucleotide primers are used toamplify a portion of SEQ ID NO:3 that can be used as a specific probe.

[0080] Other uses for the Per2 regulatory sequence include use as astarting material for generating, e.g., knockout constructs that areused to create non-human transgenic marnmals that contain a disruptionin the Per2 regulatory sequence and that are unable to express Per2.Alternatively, the Per2 regulatory sequence may be operably linked to aDNA sequence encoding a polypeptide that is not PER2 (i.e., aheterologous polypeptide).

[0081] Hybridization under stringent conditions can be used to identifynucleic acid sequences that contain a regulatory sequence of mouse PER2,or other related PER2 regulatory seqeuences. A related nucleic acidsequence has at least 50% sequence identity to mouse PER2 regulatorysequence (SEQ ID NO:3). Standard hybridization conditions are describedabove.

[0082] Circadian Proteins

[0083] The invention includes screening methods which are used toidentify compounds which can disrupt the association of mammaliancircadian proteins, e.g., the association of TIM with CRY, CRY with CRY,CRY with PER, CRY with BMAL-1, and CRY with CLOCK:BMAL-1. The inventionalso features antibodies generated against CRY, PER, and TIM proteins.These various uses require a source of CRY, TIM, PER, CLOCK, BMAL-1, andCLOCK:BMAL-1.

[0084] Circadian proteins can be isolated and purified from a naturalsource. Alternatively, the proteins can be produced recombinantly orchemically synthesized by conventional methods. Typically the proteinswill be produced recombinantly. The nucleotide and amino acid sequencesof the circadian proteins are publicly available to one skilled in theart, e.g., mouse CRY1 (Genbank accession # AB000777), mouse CRY2(Genbank Accession # AB003433), mouse TIM (Genbank accession #AF071506), mouse PER3 (Genbank accession # AF050182), CLOCK (Genbankaccesssion # AF000998) and BMAL-1 (Genbank accession # AB015203).

[0085] Methods of generating a recombinant circadian protein or arecombinant circadian fusion protein, e.g., CLOCK:GST, are well known inthe art. For example, the circadian proteins can be generated by cloningthe nucleic acid sequence encoding a circadian protein into anexpression vector, where it is operably linked to one or more regulatorysequences. The need for, and identity of, regulatory sequences will varyaccording to the type of cell in which the circadian protein sequence isto be expressed. Examples of regulatory sequences includetranscriptional promoters, enhancers, suitable mRNA ribosomal bindingsites, and sequences that terminate transcription and translation.Suitable regulatory sequences can be selected by one of ordinary skillin the art. Standard methods can be used by the skilled person toconstruct expression vectors. See, generally, Sambrook et al., 1989,Cloning—A Laboratory Manual (2nd Edition), Cold Spring Harbor Press.

[0086] Vectors useful in this invention include plasmid vectors andviral vectors. Viral vectors can be those derived from, for example,retroviruses, adenovirus, adeno-associated virus, SV40 virus, poxviruses, or herpes viruses. Once introduced into a host cell (e.g.,bacterial cell, yeast cell, insect cell, or mammalian cell), the vectorcan remain episomal, or be incorporated into the genome of the hostcell.

[0087] In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the geneproduct being expressed. For example, when a large quantity of such aprotein is to be produced, e.g., for studying the interaction of a CRYprotein with other proteins or for raising antibodies to the protein, avector capable of directing the expression of high levels of a fusionprotein (e.g., a GST fusion protein) that is readily purified may bedesirable. Alternatively, in mammalian host cells, a number ofviral-based expression systems can be utilized.

[0088] Construction of GST Fusion Proteins

[0089] In certain screening assays (see below) it may be desirable toimmobilize the circadian protein. One method of immobilizing a circadianprotein is to express the protein as a fusion protein with GST. To dothis a chimeric gene encoding a GST fusion protein can be constructed byfusing DNA encoding a circadian protein to the DNA encoding the carboxylterminus of GST (see e.g., Smith et al., Gene 67:31, 1988). The fusionconstruct can be transformed into a suitable expression system, e.g., E.coli XA90 in which expression of the GST fusion protein can be inducedwith isopropyl-β-D-thiogalactopyranoside (IPTG).

[0090] Purification of GST Fusion Proteins

[0091] After transformation of the construct into a suitable expressionsystem, induction with IPTG should yield the fusion protein as a majorconstituent of soluble, cellular proteins. The fusion proteins can bepurified by methods known to those skilled in the art, includingpurification by glutathione affinity chromatography. The purity of theproduct can be assayed by methods known to those skilled in the art,e.g., gel electrophoresis.

[0092] Binding of Circadian Proteins to Immobilized GST

[0093] GST fusion proteins can be complexed to glutathione which isattached to a matrix material, e.g., glutathione Sepharose, by methodsknown to those skilled in the art.

[0094] Antibodies

[0095] Antibodies which specifically bind to mouse or human CRY, mouseor human TIM, or mouse or human PER, or mouse or human BMAL-1 are alsoincluded in the invention. An antibody that specifically binds a mouseor human CRY, PER, TIM, or BMAL-1 is an antibody that binds only tomouse or human CRY, PER, TIM or BMAL-1 and does not bind to (i) othermolecules in a biological sample or (ii) CRY, PER, TIM or BMAL-1 ofanother organism (e.g., Drosophila or yeast).

[0096] Antibodies against mouse or human CRY, PER, TIM or BMAL-1 can beused, for example, to inhibit the interaction between these circadianproteins. Anti-CRY, -TIM or PER antibodies (e.g., monoclonal antibodies)can also be used to isolate a CRY, TIM or -PER protein using techniqueswell known in the art, such as affinity chromatography orimmunoprecipitation. The antibodies are also useful in the screeningassays described below. Compounds bound to the immunopreceipitatedprotein can then be identified.

[0097] Antibodies specific for mouse CRY, TIM, PER or BMAL-1 can beraised by immunizing a suitable subject (e.g., rabbit, goat, mouse orother mammal) with an immunogenic preparation which contains the mouseor human CRY, TIM, PER or BMAL-1 protein. An appropriate immunogenicpreparation can contain, for example, a recombinantly expressed orchemically synthesized CRY, or an immunogenic fragment thereof. Thepreparation can further include an adjuvant, such as Freund's completeor incomplete adjuvant, or similar immunostimulatory agent. Immunizationof a suitable subject with an immunogenic CRY, TIM, PER or BMAL-1preparation induces a polyclonal anti-CRY, TIM, PER or BMAL-1 antibodyresponse.

[0098] The term antibody refers to immunoglobulin molecules andimmunologically active portions of immunoglobulin molecules. Examples ofimmunologically active portions of immunoglobulin molecules includeF(ab) and F(ab′)₂ fragments, which can be generated by treating theantibody with an enzyme such as pepsin. The term monoclonal antibody ormonoclonal antibody composition refers to a population of antibodymolecules that contain only one species of an antigen binding sitecapable of immunoreacting with a particular epitope of the polypeptide.A monoclonal antibody composition thus typically displays a singlebinding affinity for the CRY, TIM or PER with which it immunoreacts.

[0099] Polyclonal anti-CRY, -TIM or -PER antibodies can be prepared byimmunizing a suitable subject with a mouse CRY, TIM or PER immunogen.The anti-CRY, -TIM or -PER antibody titer in the immunized subject canbe monitored over time by well known techniques, such as with an enzymelinked immunosorbent assay (ELISA) using immobilized polypeptide. Ifdesired, the antibody molecules directed against CRY, TIM, PER or BMAL-1can be isolated from the mammal (e.g., from the blood) and furtherpurified by well-known techniques, such as protein A chromatography, toobtain the IgG fraction.

[0100] Monoclonal antibodies can be generated by immunizing a subjectwith an immunogenic preparation containing a CRY, TIM, PER or BMAL-1. Atan appropriate time after immunization, e.g., when the anti-CRY, -TIM,-PER or BMAL-1 antibody titers are highest, antibody-producing cells areobtained from the subject and used to prepare monoclonal antibodies bytechniques well known in the art, such as the hybridoma techniqueoriginally described by Kohler et al., Nature 256:495-497, 1975, thehuman B cell hybridoma technique (Kozbor et al., Immunol Today 4:72,1983), the EBV-hybridoma technique (Cole et al., Monoclonal Antibodiesand Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) or trioma techniques.The technology for producing monoclonal antibody hybridomas is wellknown (see generally Current Protocols in Immunology (1994) Coligan etal. (eds.) John Wiley & Sons, Inc., New York, N.Y.). Briefly, animmortal cell line (typically a myeloma) is fused to lymphocytes(typically splenocytes) from a mammal immunized with a CRY, TIM, PER orBMAL-1, immunogen as described above, and the culture supernatant of theresulting hybridoma cells that screened to identify a hybridomaproducing a monoclonal antibody that binds the CRY, TIM, PER or BMAL-1.

[0101] The anti-CRY, -TIM, -PER or BMAL-1 antibody may be coupled to adetectable substance. Examples of detectable substances include variousenzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, and radioactive materials. Examplesof suitable enzymes include horseradish peroxidase, alkalinephosphatase, β-galactosidase, and acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride and phycoerythrin;an example of a luminescent material is luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin;and examples of suitable radioactive materials include ¹²⁵I, ¹³¹I, ³⁵Sand ³H.

[0102] Screening Assays

[0103] The invention encompasses methods for identifying compounds thatbind to CRY; disrupt the association of TIM:CRY, CRY:CRY, CRY:PER,CRY:BMAL-1, and CRY:CLOCK:BMAL-1; inhibit or activate the transcriptionof Per2; or positively regulate the transcription of Bmal-1. Candidatecompounds that can be screened in accordance with the invention includepolypeptides, oligopeptides, antibodies, and monomeric organiccompounds, i.e., “small molecules.”

[0104] Identification of a Compound that Binds to CRY

[0105] A useful first step to identifying a compound which disrupts theassociation between different circadian proteins (e.g., TIM:CRY,CRY:CRY, CRY:PER, CRY:BMAL-1,and CRY:CLOCK:BMAL-1) is to identify acompound that binds to CRY or another circadian protein. Once acircadian binding compound is identified, the ability of the compound todisrupt the association of different circadian proteins can be assayed.Below are a number of assays which can be used to identify a compoundwhich binds to a CRY protein, e.g., CRY1 or CRY2. The examples are notmeant to be limiting and the assays can be performed with othercircadian proteins, e.g., TIM, PER, CLOCK and BMAL-1.

[0106] Methods of identifying a compound which binds a protein ofinterest are well known in the art. In one screening method, testcompounds are evaluated for their ability to bind CRY, e.g., CRY1 orCRY2. Control reactions which do not contain the compound can beperformed in parallel. The method includes immobilizing CRY usingmethods known in the art such as binding a GST-CRY to a polymeric beadcontaining glutathione or binding a CRY protein to an anti-CRY antibodywhich is attached to a solid support. The immobilized CRY is incubatedwith a test compound for a period of time that permits binding of thetest compound to CRY. Following the incubation period, unbound testcompound is removed and bound test compound detected. For example, adetectable moiety such as a radionuclide or a fluorescent label can beattached to the compound for ease of detection. Examples of radionuclideand fluorescent labels include ¹²⁵I, ¹³¹I, ³⁵S, ³H, umbelliferone,fluorescein, fluorescein isothiocyanate, and rhodamine.

[0107] Alternatively, the screening method can involve incubating alabeled test compound, with an epitope-tagged CRY protein. Followingincubation, the ability of the test compound to bind to the CRY proteinis determined using immunoprecipitation with an antibody directedagainst the epitope tag (e.g., Flag or myc). The recovery of a labeledtest compound, e.g., a radioactive compound, followingimmunoprecipitation indicates that the test compound binds to the CRYprotein.

[0108] Display libraries can also be used to identify compounds whichbind to a CRY protein. In this approach, the test peptides are displayedon the surface of a cell or viral particle, and the ability ofparticular cells or viral particles to bind an appropriate CRY protein,e.g., CRY1 or CRY2, via the displayed product can be detected in a“panning assay” (Ladner et al., WO 88/06630).

[0109] Identifying Compounds which Disrupt the Interaction of CRY:TIM,CRY:CRY CRY:PER, CRY:BMAL-1, and CRY:CLOCK:BMAL-1

[0110] The two-hybrid expression system can be used to screen forcompounds capable of disrupting CRY:TIM, CRY:CRY, CRY:PER, CRY:BMAL-1,or CRY:CLOCK:BMAL-1 associations in vivo. In this system, a GAL4 bindingsite, linked to a reporter gene such as lacZ, is contacted in thepresence and absence of a test compound with a GAL4 binding domainlinked to a circadian protein, e.g., CRY, TIM, PER, CLOCK or BMAL-1 anda GAL4 transactivation domain linked to a circadian protein, e.g., CRY,TIM, PER, CLOCK, or BMAL-1. Expression of the reporter gene is monitoredand a decrease in said expression is an indication that the testcompound inhibits the interaction of CRY with TIM, CRY with CRY, CRYwith PER, CRY with BMAL-1, or CRY with CLOCK:BMAL-1.

[0111] Another method of identifying compounds which disrupt anassociation between circadian proteins involves the determination ofwhether the test compounds can disrupt the ability of, e.g., CRY:PER, toblock CLOCK:BMAL-1-mediated transcriptional activation. In this system,an E-box sequence linked to a reporter gene such as a luciferase gene iscontacted with a CLOCK:BMAL-1 heterodimer. Binding of the CLOCK:BAML-1heterodimer to the E-box results in expression of the reporter gene. Thesystem is then contacted with a test compound and a circadian protein(e.g., a CRY protein or a circadian protein complex, e.g., CRY:PER), andexpression of the reporter gene is monitored. Since CRY and PER blockCLOCK:BMAL-1-mediated transcription, an increase in expression of thereporter gene in the presence of the test compound as compared to theexpression in the absence of the compound indicates that the compounddisrupts the ability of CRY and PER to block CLOCK:BMAL-1-mediatedtranscription. The transcription assay can be preformed in any cell thatexpresses the necessary proteins, either naturally or recombinantly,e.g., NIH 3T3 cells, COS-7 cells, or clock neuron cells.

[0112] In yet another screening method, one of the components of theCRY:TIM, CRY:CRY, CRY:PER, CRY:BMAL-1, or CRY:CLOCK:BMAL-1 bindingcomplex is immobilized. The circadian protein can be immobilized usingmethods known in the art, such as adsorption onto a plastic microtiterplate or specific binding of a GST-fusion protein to a polymeric beadcontaining glutathione. For example, to determine a compound which bindsCRY:PER, a GST-CRY can be bound to glutathione-Sepharose beads. Theimmobilized CRY is then contacted with a labeled circadian protein towhich it binds (PER in this case) in the presence and absence of a testcompound. Unbound PER can then be removed and the complex solubilizedand analyzed to determine the amount of bound labeled PER. A decrease inbinding is an indication that the test compound inhibits the interactionof CRY with PER.

[0113] A variation of the above-described screening method involvesscreening for test compounds which are capable of disrupting apreviously-formed CRY:TIM, CRY:CRY, CRY:PER, CRY:BMAL-1, orCRY:CLOCK:BMAL-1 interaction. For example, a complex comprising CRY:PERis immobilized as described above and contacted with a test compound.The disassociation of the complex by the test compound correlates withthe ability of the test compound to disrupt or inhibit the interactionof CRY with PER.

[0114] Identifying Compounds that Activate Transcription of PER2

[0115] A screening method used to identify a compound that activates orinhibits the transcription of Per2 includes providing a cell thatincludes a Per2 regulatory sequence operatively linked to a reportergene. The Per2 regulatory sequence is preferably mammalian, e.g., mousePER2 (SEQ ID NO:3; see FIG. 8). In one example, the mouse Per2regulatory sequence is operably linked to a reporter gene such as aluciferase, a chloramphenicol acetyl transferase, a beta-galactosidase,an alkaline phosphate, or a fluorescent protein gene. A test compound isthen contacted with the cell and expression of the reporter genemonitored. An increase in expression of the reporter gene in thepresence of the test compound as compared to the expression in theabsence of the compound indicates that the compound activates Per2transcription. Alternatively, a decrease in expression of the reportergene in the presence of the test compound as compared to expression inthe absence of the test compound indicates that the compound inhibitsPer2 transcription. The transcription assay can be preformed in any cellwhich undergoes a circadian rhythm, e.g., NIH 3T3 cells, COS-7 cells, orclock neuron cells.

[0116] Identifying Compounds that Positively Regulate Expression ofBMAL-1

[0117] A screening method that uses a non-human transgenic animal whosesomatic and germ cells comprise a disrupted Per2 gene can be used toidentify a compound that regulates expression of Bmal-1. The methodincludes administering a test compound to the transgenic mouse anddetecting Bmal-1 expression. An increase in expression of Bmal-1,compared to a control non-human transgenic animal, indicates that thecompound positively regulates expression of Bmal-1. Expression of Bmal-1can be detected using any appropriate method, e.g., detecting Bmal-1mRNA levels using Northern blot analysis or BMAL-1 protein levels usinga BMAL-1 specific antibody or an activity assay.

[0118] The transgenic non-human animal used in the method describedabove includes a non-human animal that contains a disruption in the Per2gene that is sufficient to inhibit the ability of PER2 to positivelyregulate Bmal-1. A transgenic non-human animal is preferably a mammalsuch as a rat or mouse, in which one or more of the cells of the animalinclude a disruption in the Per2 gene. Other examples of transgenicanimals include non-human primates, sheep, dogs, cows, goats, chickens,amphibians, and the like. The transgenic non-human animal is one inwhich the Per2 gene has been altered, e.g., by homologous recombinationbetween the endogenous gene and an exogenous DNA molecule introducedinto a cell of the animal, e.g., an embryonic cell of the animal, priorto development of the animal. Appropriate PER2 transgenic animals whichcan be used in the method described above are known in the art, e.g.,the homozygous mPer2^(brdm1) described by Zheng et al. (Nature, 400:1667(1999)) the contents of which are incorporated herein by reference.

[0119] Modulating the Circadian Clock

[0120] Based on the discoveries described herein, it is apparent thatexpression of Bmal-1 is critical for restarting the circadian loop. Theimportance of Bmal-1 mRNA rhythm is to drive a Bmal-1 rhythm after afour to six hour delay in the circadian loop. The expression of Bmal-1makes BMAL-1 available to heterodimerize with CLOCK to drivetranscription of circadian proteins, such as Per or Cry. Thetranscription of Per or Cry restarts the cycle. Therefore, a method ofmodulating a circadian-clock controlled rhythm includes, for example,altering the endogenous expression of Bmal-1. In one example, aneffective amount of a ribozyme, or an oligouncleotide antisense toBmal-1, can be introduced into a SCN in vivo, thereby inhibitingexpression of Bmal-1 in the cell and modulating circadian-clock rhythms.

[0121] Antisense Bmal-1 nucleic acid molecules include molecules whichare complementary to a sense nucleic acid encoding a BMAL-1 protein,e.g., complementary to the coding strand of a double-stranded cDNAmolecule or complementary to a mRNA sequence. Accordingly, an antisensenucleic acid can hydrogen bond to a sense nucleic acid. Antisense Bmal-1nucleic acids can be designed according to the rules of Watson and Crickbase pairing. The antisense nucleic acid molecule can be complementaryto full length Bmal-1 mRNA, but more preferably is an oligonucleotidethat is antisense to only a portion of the Bmal-1 mRNA, e.g., part orall of the transcription start site, and/or part or all of the codingregion. An antisense oligonucleotide can be, for example, about 5, 10,15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.

[0122] The Bmal-1 antisense nucleic acid molecules are typicallyadministered to a subject such that they hybridize with or bind tocellular mRNA and/or genomic DNA encoding a protein to thereby inhibitBmal-1 expression of the protein. An example of a route ofadministration of antisense nucleic acid molecules of the inventionincludes direct injection at a tissue site. Alternatively, antisensenucleic acid molecules can be modified to target selected cells and thenadministered systemically. For example, for systemic administration,antisense molecules can be modified such that they specifically bind toreceptors or antigens expressed on a selected cell surface, e.g., bylinking the antisense nucleic acid molecules to peptides or antibodieswhich bind to clock neuron cell surface receptors or antigens. Inanother example, the antisense nucleic acid molecule is linked to TAT, aHIV leader sequence, that can target the antisense to the SCN(Lisziewicz et al., Hum Gene Ther 11:807-15, 2000).

[0123] Alternatively, an expression vector encoding BMAL-1 protein canbe introduced into a clock neuron using gene therapy methods. Forexample, methods of targeting a vector containing a Bmal-1 sequence intoan SCN include using a gene therapy vector which includes a tat sequenceoperably lined to a Bmal-1 nucleic acid sequence. Expression of TATtargets the vector to the SCN.

[0124] The gene therapy expression vector can be in the form of arecombinant plasmid, phagemid or attenuated virus in which a mammalianBMAL-1 is operably linked to an appropriate regulatory sequence.Examples of suitable viral vectors include recombinant retroviralvectors (Valerio et al., 1989, Gene, 84:419; Scharfman et al., 1991,Proc. Natl. Acad. Sci., USA, 88:462; Miller, D. G. & Buttimore, C.,1986, Mol. Cell. Biol., 6:2895), recombinant adenoviral vectors(Freidman et al., 1986, Mol. Cell. Biol., 6:3791; Levrero et al., 1991,Gene, 101:195), and recombinant Herpes simplex viral vectors. Theregulatory sequence can be the same as the endogenous regulatorysequence, or different. It can be inducible or constitutive. Suitableconstitutive regulatory sequences include the regulatory sequence of ahousekeeping gene such as the α-actin regulatory sequence, or may be ofviral origin such as regulatory sequences derived from mouse mammarytumor virus (MMTV) or cytomegalovirus (CMV).

[0125] Utility of the Compounds

[0126] Compounds found to disrupt the interaction of CRY:TIM, CRY:CRY,CRY:PER, CRY:BMAL-1, or CRY:CLOCK:BMAL-1 or bind to CRY can be used tomanipulate the circadian clock. For example, the association of PER withCRY in the cytoplasm of a clock neuron is necessary for thetranslocation of PER into the nucleus of the cell. Once PER is in thenucleus, PER has a negative feedback effect on the circadian loop, i.e.,inhibits CLOCK:BMAL-1-mediated transcription. A compound which disruptsthe ability of CRY and PER to associate in the cytoplasm would preventthe translocation of PER to the nucleus and would therefore be usefulfor blocking PER's negative feedback effect on the circadian loop.Similarly, a compound that binds to CRY is potentially useful forblocking CRY's negative feedback effect on the circadian loop.

[0127] Compounds that can modulate the transcription of the Per2 genecan be used to advance or delay restarting the circadian loop. Forexample, a compound that inhibits transcription of Per2 will inhibit thetranscription of Bmal-1. Since BMAL-1 is needed to restart the circadianloop, a compound that inhibits transcription of Per2 will inhibit therestarting of the circadian loop. Moreover, delivery of an expressionvector encoding a mammalian Bmal-1 protein to a clock neuron can also beused to manipulate the circadian rhythm and advance restarting of thecircadian loop.

[0128] A compound identified as described above is therefore useful asan agent that can reset the circadian clock. The compound can be used toprevent jet lag or facilitate resetting the clock in shift workers. Inaddition, the compound can be used to improve rhythmicity, i.e., theco-ordinated regulation of outputs from cells within the SCN. Disruptionof rhythmicity is common in the elderly and affects the ability tosleep. The compound described herein can be used to improve theinteractions between neurons to allow them to arrive at a common phaseor directly reset individual neurons to a common phase. Compounds canalso be used to alleviate circadian rhythm disorders such as winterdepression or seasonal affective disorder.

[0129] Administration

[0130] The compounds described herein can be administered to a subject,e.g., a mammal such as a human, to treat a circadian rhythm disorder,e.g., jet lag, winter depression and shift work disturbance. Thecompounds can be used to specifically advance or delay the phase ofcertain circadian rhythms. The ability of a compound to reset the clockto a specified phase will depend on the nature of the agent and itsbiological half-life.

[0131] The compound can be administered alone, or in a mixture, in thepresence of a pharmaceutically acceptable excipient or carrier (e.g.,physiological saline). Given that the different CRY and PER proteins areredundant, it is preferable that the compound administered have thespecificity to affect all members of a given family, e.g., CRY1 and CRY2(CRY protein family members) or PER1, PER2 or PER3 (PER protein familymembers). Alternatively, a combination of compounds specific for eachmember of a family can be administered.

[0132] Gene therapy vectors can be delivered to a subject by, forexample, intravenous injection or local administration (see U.S. Pat.No. 5,328,470). The pharmaceutical preparation of the gene therapyvector will typically include the gene therapy vector in an acceptablecarrier.

[0133] The excipient or carrier is selected on the basis of the mode androute of administration. Suitable pharmaceutical carriers, as well aspharmaceutical necessities for use in pharmaceutical formulations, aredescribed in Remington's Pharmaceutical Sciences (E. W. Martin), a wellknown reference text in this field, and in the USP/NF (United StatesPharmacopeia and the National Formularly). A pharmaceutical compositionis formulated to be compatible with its intended route ofadministration. Examples of routes of administration include oral,rectal, and parenteral, e.g., intravenous, intradermal, andsubcutaneous, transdermal (topical), and transmucosal, administration.Compounds which are unable to cross the blood-brain barrier areadministered locally to the SCN.

[0134] As is well known in the medical arts, dosage for any givenpatient depends upon many factors, including the patient's size, bodysurface area, age, the particular compound to be administered, sex, timeand route of administration, general health, and other drugs beingadministered concurrently. Dosages for the compounds of the inventionwill vary, but determination of optimal dosage is well within theabilities of a pharmacologist of ordinary skill.

[0135] Transgenic Animals

[0136] Based on the discovery made herein, Tim is predicted to beessential for embryonic development in animals. In order to delineatethe region(s) of Tim essential for development, the invention includesnon-human transgenic animals that have a selected region of Timdisrupted. The role of this region in embryonic development can bedetermined by analyzing homozygous embryos for developmental defects,e.g., determining cellular organization in whole embryos that are fixedand embedded in paraffin around embryonic day 7.5.

[0137] Transgenic non-human animals that have a Tim disruption are alsouseful for screening for compounds that ameliorate the developmentaldefects caused by the disruption of Tim, e.g., a test compound can beadministered to a female Tim⁺/Tim⁻ heterozygote non-human animal duringand/or subsequent to mating with a male Tim⁺/Tim⁻ 0 heterozygote of thesame species. The ability of the test compound to ameliorateTim-associated defects occurring during embryonic development can bedetermined by analyzing Tim⁻ homozygous embryos for developmentaldefects, e.g., determining cellular organization in whole embryos thatare fixed and embedded in paraffin around embryonic day 7.5.

[0138] Transgenic Tim animals which overexpress TIM are also be usefulfor studying the function and/or activity of a TIM protein in circadianrhythm. For example, transgenic non-human animals are generated where anendogenous Tim regulatory element, e.g., a promoter, is replaced with anexogenous regulatory element such that the exogenous regulatory elementdrives a higher level of expression of TIM in a cell of the transgenicanimal as compared to a non-transgenic animal. The cell is preferably aneuron. The role of TIM in circadian rhythm in the transgenic animal canbe determined by analyzing circadian rhythms in locomoter activity,e.g., rhythmic wheel turning.

[0139] As used herein, a “transgenic animal” is a non-human animal, thenucleated cells of which include a transgene. The animal is preferably amammal, e.g., a rodent such as a rat or mouse. Other examples oftransgenic animals include non-human primates, sheep, dogs, cows, goats,chickens, rabbits, amphibians, and the like. A transgene is exogenousDNA or a rearrangment, e.g., a deletion of endogenous chromosomal DNA,which is integrated into or occurs in the genome of the animal's cells.A transgene can direct the expression of an encoded gene product in oneor more cell types or tissues of the transgenic animal. Othertransgenes, e.g., a knockout, reduce expression. Thus, a transgenicanimal can be one in which an endogenous Tim gene has been altered,e.g., by homologous recombination between the endogenous gene and anexogenous DNA molecule introduced into a cell of the animal, e.g., anembryonic cell of the animal, prior to development of the animal. Theanimal can be heterozygous or homozygous for the transgene.

[0140] Intronic sequences and polyadenylation signals can also beincluded in the transgene to increase the efficiency of expression ofthe transgene. A tissue-specific regulatory sequence(s) can be operablylinked to a transgene of the invention to direct expression of a TIMprotein in particular cells. A transgenic founder animal can beidentified based upon the presence of a TIM transgene in its genomeand/or expression of TIM mRNA in tissues or cells of the animals. Atransgenic founder animal can then be used to breed additional animalscarrying the transgene. Moreover, transgenic animals carrying atransgene encoding a TIM protein can further be bred to other transgenicanimals carrying other transgenes.

[0141] TIM proteins or polypeptides can be expressed in transgenicanimals or plants, e.g., a nucleic acid encoding the protein orpolypeptide can be introduced into the genome of an animal. In preferredembodiments, the nucleic acid is placed under the control of a tissuespecific promoter, e.g., a milk or egg specific promoter, and recoveredfrom the milk or eggs produced by the animal. Suitable animals are mice,pigs, cows, goats, sheep, and chickens.

[0142] The invention also includes a population of cells from atransgenic animal, as discussed herein.

[0143] Any technique known in the art may be used to generate thetransgene non-human animals discussed herein. For a review, see Gordon,1989, Transgenic Animals, Intl. Rev. Cytol. 115:171-229 and Hogan et al.“Manipulating the Mouse Embryo” (Cold Spring Harbor Press, Cold SpringHarbor, N.Y., 1986.

[0144] Experimental Information

EXAMPLE 1

[0145] mPER Proteins Interact in Mammalian Cells

[0146] The importance of mPER:mPER interactions in the negative limb ofthe clock feedback loop was examined. Previous studies using the yeasttwo-hybrid assay showed that all of the mPERs interact with one anotherand that mPER1 and mPER2 can homodimerize (Zylka et al., Neuron21:1103-1115, 1998). No interactions were detectable between mTIM andany of the mPER proteins in the yeast system. Co-immunoprecipitationexperiments were performed in mammalian cells using epitope-taggedproteins expressed in COS7 cells.

[0147] Expression plasmids were constructed that contain full-lengthcoding regions for each mPER protein and mTIM with either a hemaglutinin(HA) or a V5 epitope tag at the carboxyl terminus. For cloning, thecoding regions of mPER2 (AF035830), mPER3 (AF050182), and mTIM(AF071506) were ligated into pcDNA 3.1 containing either an N terminalor C terminal HA tag. Full-length coding regions were amplified with PfuTurboJ (Stratagene, La Jolla, Calif.) from plasmid DNA (mPER1). Correctorientation of each construct was verified by sequence analysis. Cloneswere also transcribed and translated in vitro using TnT T7 QuickJ(Promega, Madison, Wis.) to confirm that a protein of the correct sizewas produced. Moreover, clones were transiently transfected into NIH3T3cells and into COS7 cells. Crude cell extracts were prepared, westernblotted and probed with anti-V5 or anti-HA antibodies to detectfull-length, epitope-tagged proteins.

[0148] Once the constructs were generated, COS7 cells were transientlycotransfected with expression plasmids encoding mPER3-HA and eithermPER1-V5, mPER2-VS, mPER3-V5, or mTIM-VS. Cell lysates wereimmunoprecipitated with anti-HA antibody, and the immunoprecipitatedmaterial was blotted and probed with anti-V5 antibodies to assessinteractions. Briefly, co-immunoprecipitations were performed asdescribed by Lee and colleagues (Neuron 21:857-867, 1998) with thefollowing modifications. COS7 cells (5×10⁶) were seeded in 10 cm dishesand transfected the following day with the expression plasmids describedabove. Forty-eight hours post transfection, the cells were washed twicewith PBS, homogenized in binding buffer (20 mM HEPES, pH 7.5, 100 mMKCl, 2.5 mM EDTA, 5 mM DTT, 2.5 mM PMSF, 0.05% Triton X-100, 10%glycerol, 10 μg/ml leupeptin, 10 μg/ml aprotonin) and clarified bycentrifugation. Protein concentrations were determined by the Bradfordmethod according to the manufacturer's instructions (Pierce, Iselm,N.J.). Total protein (30 μg) from the clarified supernatant was combinedwith 15 μl of protein A/G agarose beads (Santa Cruz Biotechnology, SantaCruz, Calif.) and incubated for 1 hr at 4EC to remove non-specificinteractions. The samples were centrifuged and the supernatant wasincubated for 3 hrs at 4EC with anti-HA mouse monoclonal antibodies(Babco, 1:50 dilution) and 15 μl of protein A/G agarose beads.Subsequently, the beads were washed four times (400 μl binding bufferfor 10 min. per wash), mixed with 5 μl of 4×sodium dodecyl sulfate (SDS)gel loading buffer, boiled, and centrifuged. The supernatant wasanalyzed by SDS-polyacrylamide gel electrophoresis (PAGE) and westernblotted as follows. Total protein (5 μg) from COS7 cells was extractedas described above, separated by SDS-PAGE, and transferred to anitrocellulose membrane using a semi-dry blotting apparatus. Membraneswere blocked with 5% non-fat milk. Blots were incubated with either themouse anti-HA antibody (1:10,000) or the mouse anti-V5 antibody(1:5,000) overnight at 4EC. A goat anti-mouse horseradish peroxidasesecondary antibody (1:10,000) was used in combination with enhancedchemiluminescence (NEN) to detect proteins.

[0149] Following detection of epitope-tagged proteins with one antibody,the blots were stripped in stripping buffer (62.5 mM Tris-HCl (pH 6.7),100 mM 2-mercaptoethanol, 2% SDS) at 50EC for 30 minutes. The membranewas washed extensively (20 mM Tris, pH 7.6, 137 mM NaCl, 0.05% Tween-20)then blocked again and processed for detection of the secondepitope-tagged protein.

[0150] Western blotting of cell lysates prior to immunoprecipitationshowed that all four proteins tagged with the V5 epitope were expressedat detectable levels. The co-immunoprecipitation data showed that mPER3homodimerized and heterodimerized with mPER1 and mPER2, but did notinteract at detectable levels with mTIM. When the blot was stripped andre-probed with the anti-HA antibody, similar amounts of mPER3-HA wereprecipitated in each sample. Thus, the lack of detection of anmPER3:mTIM interaction was not due to a transfection or expressionartifact. A similar pattern of interactions was obtained when thecoimmunoprecipitation experiments were performed using mPER1-HA in placeof mPER3-HA; that is, co-immunoprecipitation of the mPER proteins butnot mTIM. These results in mammalian cells confirm the findings inyeast: each mPER can homodimerize with itself or heterodimerize withanother mPER but does not detectably interact with mTIM. Our results donot rule out the possibility of biologically relevant mPER:mTIMinteractions in the mammalian clockwork. But the data do suggest thatsuch mPER:mTIM interactions must be much weaker than the strongmPER:mPER interactions found in both yeast and mammalian cells.

EXAMPLE 2

[0151] Subcellular Location of mPER3 Changes in the Presence of mPER1 ormPER2

[0152] To determine whether mPER:mPER interactions may be important forthe nuclear translocation of the mPERs and their subsequent negativefeedback on transcription, mPER:mPER interactions were examined by firstevaluating the subcellular location of the HA-and V5-epitope taggedconstructs when transfected into NIH3T3 and COS7 cells.

[0153] Immunofluorescence of epitope-tagged proteins was used to observeprotein location within cells. Briefly, cells (3×10⁵) were seeded onglass coverslips in 6-well dishes and transfected the following day asdescribed above with 1 μg of total DNA per well. Forty-eight hours aftertransfection, cells adherent to the coverslip were washed twice withphosphate buffered saline (PBS), fixed with −20EC methanol (10 min),washed, and blocked in 5% normal goat serum/0.1%Triton X-100 in PBS (1hr). Mouse anti-V5 IgG (1:500; Invitrogen, Calsbad, Calif.) or rabbitanti-HA IgG (1:200; Santa Cruz Biotechnology, Santa Cruz, Calif.) wasapplied for 1.5 hrs. Cells were washed and then incubated in the dark (1hr) with secondary antibodies. These consisted of either goatanti-rabbit IgG conjugated to Cy2 (1:200) or goat anti-mouse IgGconjugated to Cy3 (1:200; Jackson ImmunoResearch). Cells were washed,and the nuclei were stained with bisBenzimide and then mounted forfluorescence microscopy. A random population of 30-60 cells from eachcoverslip was examined by epifluorescence microscopy and the subcellulardistributions of the transfected proteins were recorded withoutknowledge of the treatment. At least three independently transfectedcoverslips were analysed. The cellular location was scored as one ofthree categories: both cytoplasm and nucleus, cytoplasm alone, ornucleus alone.

[0154] When expressed singly in NIH3T3 cells, mPER1 and mPER2 were eachfound predominantly in both cytoplasm and nucleus of individual cells(78% and 61% of transfected cells, respectively; n=3 experiments), butwere also detected in the nucleus alone (15% and 29%, respectively). Incontrast, mPER3 was mostly in cytoplasm alone (95% of transfectedcells), and mTIM was mostly nucleus alone (89%).

[0155] To determine whether co-expression promotes nuclear entry of theproteins, all possible pairwise combinations of the mPER and mTIMplasmids were co-transfected. mTIM co-expressed with any of the mPERproteins did not affect subcellular location of mTIM or the mPERproteins (p>0.05). The most obvious example of this was observed whenmPER3 and mTIM were coexpressed: mPER3 remained cytoplasmic, and mTIMremained nuclear. The inability of mTIM to influence subcellularlocation of the mPER proteins provides further evidence that mTIM doesnot interact functionally with the mPER proteins in a manner analogousto the interactions of PER and TIM in Drosophila.

[0156] When mPER3 was co-expressed with either mPER1 or mPER2, mPER3 wasdramatically redistributed from cytoplasm only to both cytoplasm andnucleus (p<0.01, n=3 experiments). mPER1 was more effective than mPER2in promoting nuclear entry of mPER3; that is, nucleus-only location wasfound in 3 times more cells with mPER1 co-transfections, compared withmPER2. The same redistribution profile was observed when the amounts ofthe mPER1 and mPER3 plasmids transfected were decreased by 75% (from 500ng to 125 ng). All of the subcellular localization experiments describedabove in NIH3T3 cells were also performed in COS7 cells with similarresults. Despite trying all possible combinations of mPER proteins withmTIM, including adding all four proteins at once, we were unable toinduce a “nucleus-only” location of mPER1 or mPER2 in >30% of NIH3T3cells. Thus, it would appear that the tested combinations do notcompletely reconstitute mPER function in NIH3T3 cells. This suggestedthat there are other clockrelevant factors important for the nucleartranslocation of the mPER proteins.

EXAMPLE 3

[0157] mPER:mPER Interactions do not Augment Inhibition ofCLOCK:BMAL-1-Induced Transcription

[0158] The ability of mPER1/2:mPER3 interactions to promote the nuclearentry of mPER3 and augment the inhibition of CLOCK:BMAL1-inducedtranscription was examined. For these studies, a luciferase reportergene assay in NIH3T3 cells was used. The reporter construct utilizes a200 bp fragment of the promoter region of the mouse arginine vasopressin(prepropressophysin) gene containing a CACGTG E box, as previouslydescribed (Jin et al., Cell 96:57-68, 1999). This reporter geneconstruct is activated by CLOCK and BMAL1 acting together on the E boxenhancer (Jin et al., supra). Briefly, luciferase reporter gene assayswere performed in NIH3T3 cells as previously described (Gekakis et al.,Science 280:1564-1569, 1998; Jin et al., supra). Cells (3×10⁵) wereseeded in six-well plates and transfected the following day. Eachconstruct contained the vasopressin promoter (10 ng) or 1.8 kb of the 5′flanking region of the mPerl, gene each cloned into pGL3 BasicJ(Promega, Madison, Wis.) (10 ng of each reporter) and CMV βgalactosidase(25 ng). cDNAs encoding Mouse CLOCK, hamster BMAL-1 and human MOP4, eachsubcloned into pcDNA3.1-V5, were each used at 250 ng per transfection.Amounts of the mPER and mTIM constructs transfected varied depending onthe experiment. The total amount of DNA per well was adjusted to 1 μg byadding pcDNA 3.1 vector as carrier. Forty-eight hours aftertransfection, cells were harvested to determine β-galactosidase activityand luciferase activity by luminometry.

[0159] Dose-response studies of inhibition of CLOCK:BMAL-1-inducedtranscription by the mPER proteins and mTIM are shown in FIG. 1. Datafrom 16 transcription assays were combined by normalizing the relativeluciferase activity values in each experiment to the activity fromCLOCK:BMAL-1 alone (set at 100%). The amounts of the mPER or mTIMexpression constructs transfected are listed (in ng) at the extremes ofthe triangles. Individual experiments were done in duplicate ortriplicate. Values are plotted as the mean %+SEM when three or moreexperiments were performed with a given amount of expression construct.All other values represent averages from two experiments.

[0160] Results showed that CLOCK:BMAL-1-induced transcription wasmaximally inhibited transfection of 250 ng of each of the mPer and mTimconstructs. Maximal inhibition reached 55-70% for each construct and wasnot substantially augmented by any pairwise transfection of the mPer andmTim constructs (at 250 ng each). As the amount of each expressionplasmid transfected was decreased, there was decreasing inhibition ofCLOCK:BMAL-1 transcription (FIG. 1). From the dose-response curves, theamount of each expression construct that was at the threshold of causingtranscriptional inhibition was identified.

[0161] Using threshold amounts of each expression construct, allpossible pairwise mPER:mPER and mPER:mTIM combinations were nextexamined to look for synergistic or additive interactions. In noinstance, however, was there observed a consistent augmentation oftranscriptional inhibition with low-dose, pairwise combinations of mPERexpression constructs or mPER plus mTIM expression constructs (n=4experiments). Co-expression experiments with low doses of mPER1 andmPER3 did show a consistent trend toward inhibition ofCLOCK:BMAL-1-induced transcription, but the effects were significant(p<0.05) in only one of three experiments.

[0162] The data hint that mPER1:mPER3 heterodimers may be functionallyrelevant for transcriptional inhibition. The endogenous expression ofthe mPer1, mPER2, mPer3, and mTim genes in NIH3T3 cells may obscurefinding a more robust inhibitory effect on transcription. Based on themodest effects of mPER:mPER interactions on nuclear localization andtranscriptional inhibition, however, it seemed more likely that therewere other factors necessary for nuclear translocation and/or retentionof the mPER proteins and for their subsequent inhibition ofCLOCK:BMAL-1-induced transcription.

EXAMPLE 4

[0163] mCry1 and mCry2 RNA Levels in the SCN and in Peripheral Clocksare Regulated by CLOCK

[0164] It was next determined if cryptochromes were involved in theCLOCK:BMAL1-driven mPer feedback loop. mCry1 and mCry2 gene expressionin wild-type and homozygous Clock mutant mice was examined, because adecrease in gene expression in Clock/Clock mice (i.e., mice homozygousfor the mutation) would place the cryptochrome genes within theCLOCK-driven feedback loop.

[0165] Northern analysis was used to examine gene expression of CRY1 andCRY2. Briefly, total RNA was extracted from tissues using the UltraspecRNA isolation reagent. Polyadenylated (polyA+) RNA was prepared usingoligotex poly dT spin columns (Qiagen, Valencia, Calfi.). PolyA+ RNA wasseparated by electrophoresis through a 1% agarose-formaldehyde gel,blotted onto GenScreenJ (New England Nuclear), and hybridized withrandom prime-labeled probe (S.A.=2×10⁶ cpm/ml). The blots werehybridized with Express HybridizationJ Solution (Clontech, Palo Alto,Calif.) and washed following the manufacturer's protocol. Probes usedwere mCry1 (nt 1081-1793 of Act. No. AB000777) and mCry2 (nt 1060-1664of Act. No. AB003433). Probe for actin was from human B-actin, purchasedfrom Clontech (Palo Alto, Calif.). Blots were exposed at −80EC to filmwith 2 intensifying screens.

[0166] Four blots were prepared from the RNA samples, with each blotconsisting of the eight time-points from one genotype and a standardlane. One microgram of polyA +RNA was loaded per lane for each genotype.Each blot was probed, stripped, then reprobed to detect mCry1, mCry2,and actin. To calculate relative RNA abundance, optical densities ofmCry1 and mCry2 hybridization were divided by densities from actinhybridization to the same blot. Normalized values were then averaged forthe two replicate blots prepared from a single set of RNA samples.Comparison across blots probed and exposed under similar conditionssuggested that the absolute level of expression of the mCry genes waslower in Clock/Clock mice than in wild-type mice. This difference inabsolute expression level was confirmed using two additional blots thatincluded selected (peak-trough) RNA samples from the two genotypesside-by-side, and were probed for both mCry1, mCry2, and actin.

[0167] mCry RNA levels in SCN are depicted in FIG. 2A. Panels depict thetemporal profiles of mCry1 RNA levels (left) and mCry2 RNA levels(right) in the SCN of wild-type mice (solid lines) and Clock/Clock mice(dashed lines). Each values is the mean+SEM of 4 animals. The horizontalbar at the bottom of the panels represents lighting cycle prior toplacement in DD; the stippled areas represent subjective day; and thefilled areas represent subjective night. Photomicrographs showedrepresentative autoradiographs of mCry1 and mCry2 gene expression fromcoronal brain sections (15 μm) at the level of the SCN from wild-type(+/+) and Clock/Clock (CZk/CZk) mice at CT 9. The brain sections wereexamined by in situ hybridization using cRNA probes as follows. Abreeding colony of mice carrying the Clock mutation was established on aBALB/c background. For studies, both males and female mice 5-15 weeks of24 age were used. Mice were housed in LD, except as noted. Animals werekilled by decapitation. Genotypes were determined using a PCRmutagenesis method, as previously described (Jin et al., supra).

[0168] Antisense and sense cRNA probes were generated from each plasmidby in vitro transcription in the presence of ³⁵S-UTP (1200 Ci/mmol).Probe for mCry1 (AB000777) was nucleotides 1081-1793 and for mCry2(AB003433) was nucleotides 1060-1664. Probe quality and size wasconfirmed by determining ³⁵S incorporation into TCA-precipitablematerial, and by gel electrophoresis and subsequent autoradiography ofthe gel.

[0169] Prehybridization, hybridization, and wash procedures wereperformed as described by Weaver. Probe (50 μl at 107 cpm/ml) wasapplied to each slide. Coverslipped slides were then incubated inhumidified chambers overnight at 55E C. Following completion of the washsteps, slides were air dried and exposed to Kodak BioMax MR film for 8days.

[0170] Densitometric analysis of hybridization intensity wasaccomplished using NIH Image software on a Macintosh computer; data areexpressed as absolute optical density values as determined bycalibration with Kodak photographic step tablet #3. ¹⁴C standardsincluded in each cassette were used to verify that the optical densityvalues measured were within the linear response range of the film.

[0171] The results showed that mCry1 RNA levels exhibited a prominentcircadian rhythm in the SCN of wild-type animals (ANOVA, p<0.05; FIG.2A). The phase of the mCry1 RNA rhythm was most similar to the phase ofthe mPER2 RNA oscillation in the SCN. In sharp contrast to wild-typemice, no mCry1 RNA rhythm was apparent in the SCN of Clock/Clock mice(ANOVA, p>0.05; FIG. 2A). Thus, the mCry1 RNA rhythm is dependent on afunctional CLOCK protein. These results are similar to the finding thatthe amplitude of RNA rhythms for each of the three mPer genes ismarkedly reduced in Clock/Clock mice (Jin et al., supra).

[0172] mCry2 RNA levels in the SCN of wild-type animals did not show acircadian rhythm (FIG. 2A; p>0.05). Interestingly, mean steady-statemCry2 RNA levels were nonetheless significantly lower in Clock/Clockmice, compared to those in wildtype controls (ANOVA, p<0.005). Thisfinding suggests that mCry2 transcription is also at least partiallydependent on a functional CLOCK protein. It is worth noting that of 5genes studied whose RNA levels do not manifest a circadian rhythm in theSCN, mCry2 is the only one in which mRNA levels in Clock/Clock animalswere observed (see Jin et al., supra). Since circadian clocks alsoappear to exist in peripheral tissues (Balsalobre et al., Cell93:929-937, 1998; Zylka et al., Neuron 20:1110, 1998b; Sakamoto et al.,J. Biol. Chem. 273:27039-27042, 1998), the temporal profiles of mCry1and mCry2 RNA levels in skeletal muscle were examined. This tissue waschosen because the three mPer genes manifest robust RNA rhythms there(Zylka et al., 1998b, supra). mCry RNA levels in skeletal muscle areshown in FIG. 2B. Autoradiograms (upper panels) illustrate Northernblots of mCry1 (3.0 kb transcript, left) and mCry2 (4.4 kb transcript,right) RNA levels at each of 8 time points in 12L:12D, with lights onfrom Zeitgeber Times (ZT) 0-12. The lower panels depict quantitativeassessment of mCry1 and mCry2 RNA levels in skeletal muscle of wildtype(solid lines) and Clock/Clock mice (dashed lines). The values are theaverage relative intensity of two replicate blots with each probe. Datawere normalized and expressed relative to hybridization intensity ofactin control probe. Data at ZT 21, ZT0/24, and ZT3 are double plotted.In contrast to the situation in the SCN, both mCry1 and mCry2 RNA levelsin muscle exhibited a daily rhythm under 12 hrs light:12 hrs dark (LD)(FIG. 2A) and a circadian rhythm under constant darkness. The peak ofthe mCry2 rhythm preceded that of mCry1 by 6 to 9 hrs, and the mCry1 RNArhythm was delayed by several hrs relative to the phase of its RNArhythm in the SCN. A phase delay between the SCN and peripheraloscillations is also observed in the RNA rhythms of the three mPer genes(Zylka et al., 1998b, supra). In skeletal muscle of Clock/Clock animals,the mCry1 RNA rhythm was dampened and phase advanced, while the mCry2RNA rhythm was abolished (FIG. 2B). For both genes, RNA levels werelower in Clock/Clock animals at all times, compared to wild-typecontrols.

[0173] Taken together, these data indicate that the transcriptionalregulation of mCry 1 and mCry2 is under CLOCK control in both the SCNand in peripheral clocks. These findings provide strong evidence thatthe mouse cryptochromes are components of the CLOCK: BMAL-1-drivenfeedback loop. Moreover, the occurrence of a CACGTG E box 300 bpupstream of the mCry1 transcription start site suggests that CLOCKdirectly participates in rhythmic mCry1 transcription through an E boxenhancer in its promoter.

EXAMPLE 5

[0174] mCRY1 and mCRY2 Block CLOCK:BMAL-1-induced Transcription inNIH3T3 Cells

[0175] The involvment of mammalian cryptochrome within the negative limbof the feedback loop was analyzed by determining whether mCRY1 and/ormCRY2 can inhibit CLOCK: BMAL-1-induced transcription. For this phase ofstudy, 14 luciferase reporter gene studies were performed in NIH3T3cells using either the vasopressin promoter (Jin et al., supra) or 1.8kb of the 5′ flanking region of the mPer1 gene subcloned into apromoterless luciferase reporter vector.

[0176] Inhibition of CLOCK:BMAL-1-mediated transcription from thevasopressin (AVP) promoter (FIG. 3A) or mPer1 promoter (FIG. 4B) bymPER1, mCRY1 and mCRY2 (250 ng each) was determined. Each value is themean+SEM of three replicates from a single assay. The results arerepresentative of three independent experiments. Dose-response curvesfor mCRY1 (Fig. cD) or mCRY2 (FIG. 3D) inhibition ofCLOCK:BMAL-1-mediated transcription from the vasopressin (AVP) promoter.Each value is the mean+SEM of three replicates from a single assay.Similar results were found in replicate experiments.

[0177] Results show that when vasopressin and mPer1 promoters were usedin the reporter vectors, mPER1 caused a maximal inhibition of 61% and30%, respectively. mCRY1 and mCRY2, on the other hand, inhibitedCLOCK:BMAL-1-induced transcription by >90% from either reporter. Thisdramatic effect on transcriptional inhibition was dose dependent foreach of the two mCRY proteins. These results indicate that mCRY1 andmCRY2 are each potent inhibitors of CLOCK:BMAL-1-mediated transcription.The mCRY-induced transcriptional inhibition must occur through direct orindirect interaction with the CLOCK:BMAL-1:E box complex because this isthe only complex common to both the vasopressin and mPer1 promoters

EXAMPLE 6

[0178] Both mCRY1 and mCRY2 are Nuclear Proteins

[0179] For the mCRY proteins to interact with the CLOCK:BMAL-1:E boxcomplex, they must be present in the nucleus. Previous studies haveshown that mCRY2 is indeed a nuclear antigen (Kobayashi et al., NucleicAcids Res. 26:5086-5092, 1998; Thresher et al., Science 282:1490-1494,1998). The situation with mCRY1 is ambiguous because previous studies ofthe endogenous protein and green fluorescent protein (GFP)-tagged mCRY1fragments indicate localization mainly in mitochondria (Kobayashi etal., supra). To determine the localization of CRY1 or CRY2, the CRYproteins were tagged at the ends of the protein with a number ofdifferent epitopes. For example, the coding regions of mCRY1 (AB000777)were ligated into the pcDNA 3.1 VS-His expression vector containingeither an N terminal or C terminal HA tag. For mCRY2, the nucleotidesequence encoding the amino terminal portion of the coding region wasnot available in GenBank (partial clone accession no. AB003433). The5′end of the mCRY2 coding region was thus cloned by 5′rapidamplification of cDNA ends. The full-length coding region was thenamplified as described above, sequenced, and deposited in GenBank asAccession Number AF156987. The constructs (FIG. 4) were transfected intoNIH3T3 cells and both their cellular localization (byimmunofluorescence) and ability to inhibit CLOCK:BMAL-1-inducedtranscription were assessed.

[0180] The results clearly showed that mCRY1 translocates to the nucleuswhen tagged with either the V5 or HA epitope. This was true when HA wasplaced at either the N-terminal or C-terminal ends, as well as whenepitope tags were placed on both ends of the protein. In each instance,the protein was nuclear and inhibited CLOCK:BMAL1-induced transcriptionby >90%. Interestingly, when enhanced (E)GFP was fused to either end ofmCRY1, immunofluorescence was found diffusely throughout the cell andthere was no transcriptional inhibition. The same diffuse staining andlack of transcriptional inhibition was found with EGFP alone. When EGFPwas fused to an N-terminal fragment of mCRY1 containing a putativesignal sequence for transport into mitochondria, the cellular locationwas mainly cytoplasmic, punctate and appeared to be in mitochondria.Using a specific anti-mCRY1 antibody, endogenous mCRY1 protein was shownto be nuclear in non-transfected NIH3T3 cells and in SCN. Thus, mCRY1 isnormally a nuclear protein and that GFP fused to CRY alters the locationof the native protein by changing its conformation. mCRY2-V5 was foundin the nucleus, consistent with previous findings (Kobyashi et al.,supra; Tresher et al., supra), and the tagged protein inhibitedCLOCK:BMAL-1-induced transcription by >90%.

EXAMPLE 7

[0181] mCRY1 and mCRY2 Directly Interact with the mPER Proteins andTranslocate them into the Nucleus

[0182] To evaluate the potential for protein: protein interactionsbetween the mCRY and mPER families, co-immunoprecipitation usingepitope-tagged proteins was utilized.

[0183] COS7 cells co-transfected with expression plasmids encoding mCRY1-HA and either mPER1-V5, mPER2-VS, mPER3-V5, or mTIM-V5 expressed eachV5-tagged protein prior to immunoprecipitation. Immunoprecipitation withthe HA antibody and analysis of the immunoprecipitated material withanti-V5 antibodies indicated the presence of heterodimeric interactionsbetween mCRY1 and each of the mPER and mTIM proteins. There was nointeraction between mCRY1 and βgalactosidase which served as aspecificity control. Co-immunoprecipitation experiments using mCRY2-HAinstead of mCRY 1-HA similarily showed the presence of heterodimericinteractions between mCRY2 and each of the mPER and mTIM proteins.

[0184] Having shown that mCRY:mPER heterodimers could exist, the abilityof such interactions to translocate the mPER proteins to the nucleus wasdetermined. In marked contrast to the lack of effect of any pairwisecombination of mPER:mPER or mPER:mTIM interactions to translocate mPER1and mPER2 to the nucleus, each mCRY protein profoundly changed thelocation all three mPER proteins in NIH3T3 and COS7 cells. This was mostapparent for mPER1 and mPER2 which were almost entirely nuclear afterco-transfection with either mCRY1 or mCRY2. Curiously, each mCRY proteinchanged mPER3 from mainly cytoplasm only (>80%) to both cytoplasm andnucleus (>80%) to a degree similar to that induced by co-transfection ofmPER3 with mPER1. When mPER3 was co-transfected with mPER1 and eithermCRY1 or mCRY2, however, each of the three protein combinations changedmPER3's location from 13-20% nucleus only to predominantly nucleus only(54-68% of transfected cells). Co-transfection of either mCRY1 or mCRY2with mTIM did not change the predominantly nucleus only location (>90%of transfected cells) of any of the three proteins.

[0185] These data indicate that the mCRY proteins can heterodimerizewith the mPER proteins and mTIM. The mCRY:mPER interactions mimic the invivo situation where the interaction of mCRY and mPER results in thealmost complete translocation of mPER1 and mPER2 to the nucleus.Moreover, trimeric interactions among the mPER and mCRY proteins appearnecessary for complete nuclear translocation of mPER3. The data alsosuggest that the nuclear translocation of the mPER proteins is dependenton mCRY 1 and mCRY2. The mCRY proteins, however, appear to be able totranslocate to the nucleus independent of the mPERs. Even with massiveoverexpression of mCRY proteins in cell culture they are always >90%nuclear.

EXAMPLE 8

[0186] mCRY1 and mCRY2 Levels Express Synchronous Circadian Rhythms inthe SCN

[0187] If nuclear entry of mPER1 and mPER2 is dependent on the mCRYproteins as suggested by the cell culture experiments, then similarilysynchronous circadian oscillations of endogenous mCRY1 and mCRY2 levelsin the nuclei of SCN neurons might be expected. To determine this theoscillations of endogenous CRY in neurons was determined. Briefly, miceentrained to a schedule of 12L:12D were transferred to constant dim redlight. Circadian Time (CT) was initially defined relative to predictedlights-off (CT12), and on the day of sampling was confirmed by thecoincident onset of group activity, as monitored by passive infra-redmovement detectors. After 20 (CT8) to 42 (CT6) hours in constant dim redlight, mice were killed with an anesthetic overdose, and perfused (4%paraformaldehyde). Brains were removed, post-fixed, transferred tocryoprotectant buffered sucrose solution (20%) and then sectioned on afreezing microtome. Alternate freefloating sections (40 μm) wereincubated with affinity purified anti-mCRY1 or anti-mCRY2 (both at 0.5μg/ml) primary sera (Alpha Diagnostic International). The sera wereraised against synthetic peptides corresponding to specific sequencesclose to the C-terminals of the mCRY1 (26 amino acids) and mCRY2 (22amino acids)proteins. To test for specificity of the sera, some SCNsections were incubated with affinity purified sera to which syntheticpeptide (10 μg/ml) had been added. Immunoreaction was visualised byavidin-biotin/peroxidase in conjunction with diaminobenzidine chromogen(Vector Labs, Peterborough, U.K.). Counts of the number ofimmunoreactive nuclear profiles in the SCN were made using an imageanalysis system as described previously.

[0188] Immunocytochemical analysis of mCRY1 and mCRY2 in the brains ofmice sampled at Zeitgeber Time (ZT) 15 (3 h after lights off) identifiedthem both as nuclear antigens in the SCN and elsewhere, includingpiriform cortex (mCRY2) and hippocampus (mCRY1, mCRY2). The majority ofSCN neurons appeared to be immunoreactive for the antigen tested, andthe immunoreactivities were specific, being blocked by pre-incubationwith the peptide (10 μg/ml) used to raise the respective serum. Incontrast, the SCN from animals sampled at ZT3 contained very fewmCry1-or mCRY2-immunoreactive nuclei, and those which were evident werelocated in a dorso-lateral position comparable to that reported formPER1 immunoreactive nuclei at this phase. Rhythmic expression of mCRY1and mCRY2 was sustained under free-running conditions, with low levelsat Circadian Time (CT)2 and high expression throughout the SCN at CT14was observed. Quantitative analysis of the number of immunoreactivenuclei in the SCN sampled at 2 h intervals over 24 h in DD showed aclear circadian variation. The abundance of both proteins was low in theearly subjective day, rising in later subjective day to peak atCT12-CT16. There was a progressive decline during subjective night tobasal counts at CT24. This temporal profile of mCRY1 andmCRY2-immunoreactivity in the SCN is directly comparable with thatobserved for mPER1 and mPER2, indicative of a synchronous nuclearaccumulation of these proteins in the SCN.

[0189] In contrast, expression of mCry1- and mCRY2-immunoreactivity inother areas did not exhibit appreciable circadian variation, consistentwith the constitutive expression of mPER proteins in brain sites outsidethe SCN.

[0190] These in vivo data, in conjunction with our cell culture data,strongly suggest that the mCRY proteins are the dominant movers of themPER1 and mPER2 proteins from cytoplasm to nucleus. We do not yet knowthe temporal pattern of mPER3 immunoreactivity in the SCN, but we haveno reason to believe it will be any different from that found for mPER1and mPER2.

EXAMPLE 9

[0191] Dissociation Between the Inhibitory Effects of the mPER Proteinsand the mCRY Proteins on Transcription

[0192] By varying the amounts of mPER and mCRY plasmids inco-transfection experiments, we have observed at best additive effectsof pairwise combinations of mPER with mCRY proteins on the inhibition ofCLOCK:BMAL-1-mediated transcription. Although these studies in cellculture are confounded by the endogenous expression of the mPer1, mPER2,mPer3, mTim, mCry1 and mCry2 genes in the cell lines used, the lack ofsynergism of pairwise combinations on transcriptional inhibitionsuggested that the mPER and mCRY proteins have independent effects onthe transcriptional machinery. To examine this in more detail, the factthat MOP4:BMAL-1-heterodimers also activate transcription via a CACGTG Ebox was exploited (Hogenesch et al., Proc. Natl. Acad. Sci. USA95:5474-5479, 1998).

[0193] CLOCK, MOP4, and BMAL-1 alone or in pairwise combinations weretested for transcriptional activation (FIG. 8A). Significanttranscriptional activation was seen only when CLOCK and BMAL-1 (10-foldincrease) or MOP4 and BMAL 1 (37-fold increase) were co-expressed.Transcriptional activation was dependent on the E-box, because notranscriptional activation was detected when the vasopressin promoterwith a mutated E-box was used. The greater levels of transcriptionalactivation with MOP4:BMAL-1 than with CLOCK:BMAL-1 appeared due to muchhigher levels of MOP4 protein expression compared with CLOCK based onwestern blot analysis of epitope tagged proteins.

[0194] Each mPER alone, mTIM, or each mCRY alone was tested for itsability to inhibit MOP4:BMAL-1-induced transcription. Even though eachmPER protein can inhibit CLOCK:BMAL-1-induced transcription, the mPERproteins (500 ng of each plasmid) did not affect MOP4:BMAL-1-inducedtranscription (FIG. 5B). When the amount of MOP4 was reduced so that therelative luciferase values were equal to those seen with CLOCK andBMAL-1 activation, the mPER expression plasmids were still unable toinhibit transcription. In contrast to the lack of inhibition of the mPERproteins, mTIM (at 500 ng) was able to inhibit MOP4:BMAL-1-inducedtranscription by about 40% (FIG. 5; p>0.01). Combinations of each mPERand the mTIM expression plasmids, or pairwise combinations of mPERexpression plasmids did not inhibit more effectively than when the mTIMplasmid was transfected alone. Remarkably, each mCRY protein (250 ngeach) abrogated MOP4:BMAL-1-mediated transcription (FIGS. 5C and 5D).

[0195] These data suggest that the mPER proteins have their action onCLOCK, perhaps as mPER:mCRY heterodimers, while the mCRY proteins appearcapable of interacting directly with either BMAL-1 or the CACGTG E box.It is worth noting that MOP4 does not appear to play a major role incircadian function, as its RNA is not detectably expressed in the SCN ofeither wild-type or Clock-mutant mice.

EXAMPLE 10

[0196] Bmal1 RNA Rhythm in Clock/Clock Mutant Mice

[0197] BMAL-1 RNA rhythm was first documented in mouse SCN usingquantitative in situ hybridization (Jin et al., Cell 96:57 (1999)) withan antisense riboprobe that recognizes the two major Bmal1 transcriptsin the SCN (Yu et al., Biochem. Biophys. Res. Commun. 260:760 (1999)).Wild-type mice exhibited a robust circadian rhythm in Bmal1 RNA levels,with low levels from circadian time (CT) 6-9 and peak levels from CT15-18.

[0198] The phase of the Bmal1 rhythm is opposite that of the mousePer1-3(mPer1-3) RNA rhythms (Zylka et al., Neuron 20:1103 (1998); Oishiet al., Biochem. Biophys. Res. Commun. 268:164 (2000); Honma et al.,Biochem. Biophys. Res. Commun. 250:83 (1998)). In addition to drivingrhythmic transcription of the mPer and mCry genes (Jin et al., Cell 96:57 (1999); Kume et al., Cell 98:193 (1999)), it seemed possible thatCLOCK:BMAL1 heterodimers might simultaneously negatively regulate Bmal1gene expression, similar to a proposed model of clock gene regulation inDrosophila. If CLOCK:BMAL1 heterodimers are negatively regulating Bmal1gene expression and if the mutant CLOCK protein is ineffective in thisnegative transcriptional activity, then Bmal1 RNA levels should beelevated and less rhythmic in homozygous Clock mutant mice. Compared towild-types, however, Clock/Clock animals expressed a severely dampenedcircadian rhythm of Bmal1 RNA levels in the SCN (significant differencebetween genotypes; ANOVA, P<0.001) (FIG. 9). Trough Bmal1 RNA levels didnot differ between Clock/Clock mice and wild-types. The peak level ofthe RNA rhythm in homozygous Clock mutant mice was only ≈ 30% of thepeak value in wild-types. A similar blunting of the Bmal1 RNA rhythm inthe SCN of Clock/Clock mice has been reported by others (Oishi et al.,Biochem. Biophys. Res. Commun. 268:164 (2000)).

[0199] The temporal profile of Clock RNA levels was examined in the SCNof Clock/Clock mutant animals, since it has been reported that Clock RNAlevels (assessed by Northern blot analysis) are decreased in the eye andhypothalamus of Clock/Clock mutant mice (King et al., Cell 89:641(1991)). Consistent with previous reports (Tei et al., Nature 389:512(1997); Shearman et al., Neuroscience 89:387 (1999) Clock RNA levels didnot manifest a circadian oscillation in mouse SCN. Surprisingly, ClockRNA levels in the SCN of Clock/Clock mutant mice were not significantlydifferent from those in the SCN of wild-type animals (FIG. 10; ANOVA,P>0.05). Thus, the Clock mutation appears to alter regulation of Bmal1gene expression in SCN, but not the regulation of the Clock gene itself.Clock expression may be decreased in other hypothalamic regions.

[0200] The low levels of Bmal1 RNA in the SCN of homozygous Clock mutantanimals show that CLOCK is not required for the negative regulation ofBmal1. Instead, these data indicate that CLOCK is actually necessary forthe positive regulation of Bmal1. The positive effect of CLOCK on Bmal1levels is probably indirect and may occur via the mPER and/or mCRYproteins, which are expressed in the nucleus of SCN neurons at theappropriate circadian time to enhance Bmal1 gene expression (Kume etal., Cell 98:193 (1999); Field et al., Neuron 25:437 (2000)). Inaddition, the mPer1-3 and mCry1-2 RNA oscillations are alldown-regulated in Clock/Clock mutant mice (Jin et al., Cell 96:57(1999); Kume et al., Cell 98:193 (1999)). Reduced levels of the proteinproducts of one or more of these genes may lead to the reduced levels ofBmal1 in the mutant mice, through loss of a positive drive on Bmal1transcription.

EXAMPLE 11

[0201] Bmal1 and mCry1 RNA Rhythms in mPER2^(Brdm1) Mutant Mice

[0202] Homozygous mPER2^(Brdm1) mutant animals have depressed mPer1 andmPER2 RNA rhythms (Zheng et al., Nature 400:167 (1999)). The Bmal1rhythm in homozygous mPER2^(Brdm1) mutants was examined to determinewhether the positive drive on the Bmal1 feedback loop might come fromthe mPER2 protein. The effects of this mutation on the mCry1 RNA rhythmwere also examined.

[0203] The temporal profiles of gene expression were analyzed at sixtime points over the first day in DD in homozygous mPER2^(Brdm1) mutantmice and wild-type littermates. The Bmal1 RNA rhythm expressed in theSCN of wild-type animals was substantially altered in the SCN of mutantmice (ANOVA, P<0.05)(FIG. 11). Trough RNA levels did not differ betweenwild-type and mutant animals, but the increase in Bmal1 RNA levels wasadvanced and truncated in the mutants, compared to the wild-type rhythm.

[0204] The mCry1 RNA rhythm was also significantly altered. In the SCNof mPER2^(Brdm1) mutant mice (ANOVA, P<0.0001)(FIG. 12), the peak levelsof the mCry1 RNA rhythm were suppressed by ≈50%, as reported for mPer1and mPER2 RNA rhythms in this mouse line (Zheng et al., Nature 400:167(1999)).

[0205] These data suggest that maintenance of a normal Bmal1 RNA rhythmis important for the positive transcriptional regulation of the mPer andmCry feedback loops. Thus, rhythmic Bmal1 RNA levels may drive rhythmicBMAL 1 levels which, in turn, regulate CLOCK:BMAL1-mediatedtranscriptional enhancement in the master clock. Indeed, mPer1, mPER2,and mCry1 RNA rhythms are all blunted in the SCN of mPER2^(Brdm1) mutantmice, in which the Bmal1 rhythm is also blunted. In addition, thehomozygous mPER2^(Brdm1) mutation is associated with a shortenedcircadian period and ensuing arrhythmicity in constant darkness (DD).

[0206] These data, along with the fact that Clock RNA levels areunaltered in the SCN of homozygous mPER2^(Brdm1) mutants (Zheng et al.,Nature 400:167 (1999), also provide evidence that mPER2 is a positiveregulator of the Bmal1 RNA rhythm. This effect may be unique to mPER2.For example, the diurnal oscillation in mPer2 RNA is not altered in theSCN of mPer1-deficient mice, and mPer1, mPer2, and Bmal1 RNA circadianrhythms are not altered in the SCN of mPer3-deficient mice. Moreover,circadian rhythms in behavior are sustained in mice deficient in eithermPer1 or mPer3.

EXAMPLE 12

[0207] mCRY-Mediated Nuclear Translocation of mPER2 is PAS-Independent

[0208] There are at least two ways that the mPER2^(Brdm1) mutation couldalter the positive drive of the clock feedback loops. The mutation coulddisrupt mPER:mCRY interactions important for the synchronousoscillations of their nuclear localization and/or alter the protein'sability to interact with other proteins (e.g., transcription factors).We examined whether the PAS domain is necessary for functionallyrelevant mPER2:mCRY interactions, using immunofluorescence ofepitope-tagged proteins in COS-7 cells. Briefly, COS-7 cells (3×10⁵)were seeded on glass coverslips in 6-well dishes and transfected withLipofectamine Plus™ (Gibco BRL) with 0.5 ug of total DNA per well.Forty-eight hrs after transfection, cells were processed as described(Sangoram et al., Neuron 21:1101 (1998)). A random population of 30-60cells from each covership was examined by epiflourescence microscopy andthe subcellular distribution of expressed proteins was recorded withoutknowledge of treatment. At least three independently transfectedcoverslips were analyzed.

[0209] Coexpression of mPER1 or mPER2 with either mCRY1 or mCRY2 inCOS-7 cells translocates >90% of mPER1 and mPER2 into the nucleus (Kumeet al., Cell 98:193 (1999)). To determine whether the PAS domain ofmPER2 is required for this translocation an mPER2 fragment containingresidues 1-337 of PER2 (mPER2¹⁻³³⁷), which includes the PAS domain, wasexamined in COS-7 cells. mPER2¹⁻³³⁷ was localized to both cytoplasm andnucleus (89% of transfected cells)(FIG. 13) and the localization was notchanged by co-expression with mCRY1. Co-expression of mPER2³³⁸⁻¹²⁵⁷ withmCRY1, however, dramatically changed the cellular location of themPER2³³⁸⁻¹²⁵⁷ fragment from cytoplasm only (12%) to nucleus only (85%).Co-expression of mPER2^(Brdm1) (missing residues 348-434) with mCRY1also moved mutant mPER2^(Brdm1) into the nucleus, from cytoplasm only(100% when expressed alone) to predominantly both cytoplasm and nucleus(81%) when co-expressed with mCRY1 (FIG. 13). The same patterns ofcellular localization were found when mCRY2 was co-expressed with thesemPER2 constructs instead of mCRY1. Thus, functional mPER2:mCRYinteractions are not mediated through the PAS domain. Similarly, the PASdomain was not important for the mCRY-mediated nuclear translocation ofmPER1 in COS-7 cells.

[0210] The data show mPER:mCRY interactions necessary for nucleartransport of the mPER1 and mPER2 proteins occur through domains outsidethe PAS region. Thus, the PAS domain of an mPER2:mCRY heterodimer mightbe free to bind to an activator (e.g., transcription factor) and shuttleit into the nucleus to activate Bmal1 transcription. Alternatively, oncein the nucleus, mPER2:mCRY heterodimers or mPER2 monomers couldcoactivate Bmal1 transcription through a PAS-mediated interaction with atranscription factor (Glossop et al., Science 286:766 (1999)). mPER2itself does not possess a DNA binding motif (Shearman et al., Neuron19:1261 (1997)).

EXAMPLE 13

[0211] Bmal1 RNA Levels in Mice Lacking mCry1 and mCry2

[0212] The tonic mid-to-high mPer1 and mPer2 RNA levels inmCRY-deficient mice (van der Horst et al., Nature 398:627 (1999) suggestthat CLOCK:BMAL1 heterodimers might be constantly driving mPer1 andmPer2 gene expression in the absence of transcriptional inhibition bythe mCRY proteins. To examine whether Bmal1 RNA levels would also bemodestly elevated, Bmal1 RNA levels in the SCN of mCRY-deficient micewere compared to those in the SCN of wild-type mice of the same geneticbackground at CT 6 and at CT 18 on the first day in DD. ThemCRY-deficient (double mutant) colony of mice had a C57BL/6 X 129 hybridbackground, and wild-type controls were of the same genetic background(van der Horst et al., Neuroreport 10:3165 (1999)). Sex ratios of maleand female mice were balanced across time points. We also examined ClockRNA levels in these animals.

[0213] In wild-type animals, the typical circadian variation in Bmal1RNA levels was apparent with high levels at CT 18 and low levels at CT 6(P<0.001)(FIG. 14). In mCry-deficient mice, on the other hand, Bmal1 RNAlevels were low at both circadian times (P>0.05)(FIG. 15). Clock RNAlevels did not differ as a function of circadian time or genotype(P>0.05)(FIG. 15).

[0214] The unexpectedly low Bmal1 gene expression in the SCN ofmCry-deficient mice suggests that the Bmal1 feedback loop is disruptedin the mutant animals, with a resultant non-functional circadian clock.Nevertheless, enough Bmal1 gene expression and protein synthesis occursfor heterodimerization with CLOCK so that, without the strong negativefeedback normally exerted by the mCRY proteins, mPer1 and mPer2 geneexpression is driven sufficiently by the heterodimer to giveintermediate to high RNA values (depending on RNA stability).

EXAMPLE 14

[0215] mPER1 and mPER2 Localization in mCry-Deficient Mice

[0216] The mid to high mPer1 and mPer2 RNA levels in the SCN ofmCry-deficient mice, and simultaneous low Bmal1 levels, suggests thatmPER1 and mPER2 proteins may not be exerting much positive or negativeinfluence on the core feedback loops. To test this, immunocytochemistrywas used to determine whether mPER1 and mPER2 were tonically expressedin the nuclei of SCN cells in mCry-deficient mice, since nuclearlocation is necessary for action on transcription (Kume et al., Cell98:193 (1999); Field et al., Neuron 25:437 (2000)).

[0217] mPER1 immunoreactivity exhibited a robust rhythm of nuclearstaining in the SCN of wild-type mice, with high values at CT 12(328±3.5, mean±SEM of positive nuclei per 30 μm section, n=3) andsignificantly lower values at CT 24 (54±5, n=3; P<0.01). These valuesare very similar to those previously reported in other strains of mice(Field et al., Neuron 25:437 (2000)).

[0218] The pattern of mPER1 immunoreactivity in the SCN ofmCry-deficient mice was quite different, however. mPER1 immunoreactivitywas detected in the nucleus of a similar number of SCN neurons at eachof the two circadian times (CT 12, 140±9, n=3; CT 24, 152±21, n=3), andthe counts at each time were at ≈40% of those seen at peak (CT 12) inwild-type animals.

[0219] The double mCry mutation also altered the sub-cellulardistribution of mPER1 staining in the SCN. In wild-type mice, mPER1staining viewed under contrast interference was clearly nuclear with avery condensed immunoreaction and a clear nucleolus. The neuropil of theSCN in wild-types was devoid of mPER1 immunoreactivity. In the SCN ofmCry-deficient animals, mPER1 staining was clearly nuclear, but thenuclear profiles were less well defined and less intensely stained, andperinuclear, cytoplasmic immunoreaction could be observed. In addition,the neuropil staining for mPER1 was higher in mCry deficient mice,although dendritic profiles were not discernible. In the same brains,the constitutive nuclear staining for mPER1 normally seen in thepiriform cortex was not altered in mCry-deficient animals.

[0220] mPER2-immunoreactivity also exhibited a robust rhythm of nuclearstaining in the SCN of wild-type mice, with high counts at CT 12(371±11, n=3) and significantly lower counts at CT 24 (31±3, n=3;P<0.01), similar to that previously reported in another strain (Field etal., Neuron 25:437 (2000)). In striking contrast, the pattern ofmPER2-immunoreactivity in the SCN of mCry-deficient mice wasdramatically altered. There were extremely few mPER2-immunoreactivecells in the SCN of mCry-deficient animals at either circadian time (CT12, 12±1, n=3; CT 24, 8±2, n=3).

[0221] In the wild-type mice, the mPER2 staining profiles were clearlynuclear, with well-defined outlines and nucleoli devoid of reactionproduct. In the few mPER2-immunoreactive cells in the SCN ofmCry-deficient mice, low level mPER2 staining was observed in thenucleus, but the profiles were poorly defined and low intensityperinuclear staining could also be observed. As for mPER1, genotype hadno discernible effect on nuclear mPER2 immunoreactivity in the piriformcortex, although there was evidence of a low level of perinuclearimmunoreactivity for mPER2 in piriform cortex of mCRY-deficient mice.

[0222] The marked reduction of mPER2 staining in the SCN ofmCry-deficient animals suggests that the mCRY proteins are eitherdirectly or indirectly important for mPER2 stability, as mPER2 RNAlevels are at tonic intermediate to high levels in mCry-deficient mice,similar to those found for mPer1 RNA levels (Okamura et al., Science286:2531 (1999)). It seems unlikely that our assay is incapable ofdetecting mPER2 in the cytoplasm of mCry-mutants, since the PER2antibody can detect cytoplasmically localized antigen in SCN cells(Field et al., Neuron 25:437 (2000)).

[0223] The low levels of mPER2 immunoreactivity in the SCN ofmCry-deficient mice, in conjunction with tonically low Bmal1 RNA levels,is consistent with an important role of mPER2 in the positive regulationof the Bmal1 loop. Since mPER1 is present in SCN nuclei inmCry-deficient mice, yet Bmal1 RNA is low, it appears likely that mPER1likely has little effect on the positive regulation of the Bmal1feedback loop or negative regulation of the mPer1-3 cycles.

[0224] The immunohistochemical data also indicate that mPER1 and mPER2can each enter the nucleus even in the absence of mCRY:mPERinteractions. mPER1 is expressed in the nucleus of SCN neurons frommCry-deficient mice, and both mPER1 and mPER2 are constitutivelyexpressed in the nucleus of cells in the piriform cortex ofmCry-deficient animals. The phosphorylation state of mPER1 dictates itscellular location in the absence of mPER1:mCRY interactions, since itsphosphorylation by casein kinase I epsilon leads to cytoplasmicretention in vitro. Thus, the nuclear location of both mPER1 and mPER2in vivo may depend on several factors, including interactions with mCRYand other proteins and their phosphorylation.

EXAMPLE 15

[0225] mCRY-Induced Inhibition of Transcription

[0226] The intermediate to high levels of mPer1 and mPER2 geneexpression throughout the circadian day in mCry-deficient mice (Okamuraet al., Science 286:2531 (1999); Vitaterna et al., Proc. Natl. Acad.Sci. USA 96:12114 (1999)) is consistent with a prominent role of themCRY proteins in negatively regulating CLOCK:BMAL1-mediatedtranscription, as in vitro data have suggested (Kume et al., Cell 98:193(1999)). The endogenous expression of the mCry1, mCry2, and mPer1-3genes in mammalian cell lines, however, has obscured rigorous in vitroanalysis of the mechanism. Therefore, an insect cell line, Schneider(S2) cells, a Drosophila cell line that expresses cycle (the DrosophilaBmal1) but not per, Tim, and clock (Saez et al., Neuron 17:911 (1996);Darlington et al., Science 280:1599 (1998)), was used to study thenegative regulation of mCRY1 and mCRY2 on E box-mediated transcriptionwith a luciferase reporter that consists of a tandem repeat of theDrosophila per E box (CACGTG) and flanking nucleotides fused to hsp70driving luciferase (Darlington et al., Science 280: 1599 (1998)).Briefly, S2 cells were transfected with Cellfectin™ (Gibco BRL). Eachtransfection consisted of 10 to 100 ng of expression plasmid withindicated inserts in pAC5.1-V5, 10 ng luciferase reporter, and 25 ng ofβ-gal internal control plasmid (driven by baculovirus immediate-earlygene, ie-1 promoter). Total DNA for each transfection was normalizedusing pAC5.1-V5. Cells were harvested 48 hrs after tranfection.Luciferase activity was normalized by determining luciferase:β-galactivity ratios and averaging the values from triplicate wells.

[0227] Since S2 cells express endogenous cyc, transfection with dclockalone caused a large increase in transcriptional activity (265-fold), asdescribed (Darlington et al., Science 280: 1599 (1998)). As for dCRY(Ceriani et al., Science 285:553 (1999)), this activation was notinhibited by either mCRY1 or mCRY2. When co-transfected, mCLOCK andsyrian hamster (sh)BMAL1 heterodimers induced a large increase intranscriptional activity (1744-fold) that was reduced by >90% by mCRY1or mCRY2 (FIG. 16). Moreover, cotransfection of shBmal1 and human(h)Mop4, but not transfection of hMop4 alone, similarly caused a largeincrease in transcriptional activity in S2 cells (539-fold), like thatpreviously found for hMOP4:shBMAL1 heterodimers in mammalian cells(Hogenesch et al., Proc. Natl. Acad. Sci. USA. 95:5474 (1998); Kume etal., Cell 98:193 (1999)). hMOP4:shBMAL1-mediated transcription was alsoblocked by either mCRY1 or mCRY2 (FIG. 16). The mCLOCK:shBMAL1- andhMOP4:shBMAL1-induced transcription in S2 cells was dependent on anintact CACGTG E box, because neither heterodimer caused an increase intranscription when a mutated E box reporter was used in thetranscriptional assay. Immunofluorescence of epitope-tagged mCRY1 ormCRY2 expressed in S2 cells showed that each was >90% nuclear inlocation, as in mammalian cells (Kume et al., Cell 98:193 (1999)).

[0228] These data indicate that mCRY1 and mCRY2 are nuclear proteinsthat can each inhibit mCLOCK:shBMAL1-induced transcription independentof the mPER and mTIM proteins and of each other. The results also showthat the inhibitory effect is not mediated by the interaction of eithermCRY1 or mCRY2 with the E box itself, since E box-mediated transcriptionwas not blocked by the mCRY proteins when transcription was activated bydCLOCK:CYC heterodimers. It thus appears that the mCRY proteins inhibitmCLOCK:shBMAL1-mediated transcription by interacting with either or bothof the transcription factors, since a similar inhibition was found withhMOP4:shBMAL1-induced transcription. The system was performed asdescribed in Gekakis et al. (Science 270:811 (1995)). Yeast two-hybridassays revealed strong interactions of each mCRY protein with mCLOCK andshBMAL1. Weaker interactions were detected between each mCRY protein andhMOP4. This is further evidence of functionally relevant associations ofeach mCRY protein with each of the three transcription factors (Griffinet al., Science 286:768 (1999)).

[0229] Next it was determined whether the mCRY-induced inhibition oftranscription was through interaction with CLOCK and/or BMAL1. Sinceneither mCRY1 or mCRY2 inhibited dCLOCK:CYC mediated transcription, theability of each to inhibit dCLOCK:shBMAL1-mediated transcription wasexamined. This aspect of study could not be examined in S2 cells,because of the strong activation induced by transfecting dclock alone inS2 cells where there is strong endogenous cyc expression. Briefly,luciferase reporter gene assays were performed in COS-7 cells asdescribed (Jin et al., Cell 96:57 (1999)). mCRY1 and mCRY2 completelyinhibited mCLOCK:shBMAL1- and hMOP4:shBMAL1-induced transcription inCOS-7 cells (FIG. 17, Left and Middle, respectively), while thecryptochromes did not inhibit dCLOCK:shBMAL1-mediated transcription bymore than 20% (FIG. 17). Thus, mCRY inhibits mCLOCK:shBMAL1-inducedtranscription through interaction with either mCLOCK alone or through anassociation with both mCLOCK and BMAL1 in a multiprotein complex.Unfortunately, the examination of the inhibition of mCLOCK:CYCheterodimers was not possible, because co-transfection of mClock and cycdid not activate transcription in either insect cells or mammaliancells, even though strong interactions between mCLOCK and CYC weredetected in yeast.

EXAMPLE 16

[0230] Identifying a Role for Mouse Tim

[0231] To delineate potential functions for mTim, the gene was disruptedby targeted mutagenesis. A targeting vector was designed from a 15 kbgenomic clone in which a portion of the gene was replaced with a PGK-Neocassette; this deletion-insertion disrupts mTIM after codon 178 (of1197). Homologous recombination of the targeted allele was obtained in129/Sv J1 embryonic stem cells, and two clones were microinjected intoC57BL/6 mouse blastocysts. Chimeric offspring were mated and germlinetransmission was obtained.

[0232] When heterozygous animals were crossed, the resulting litterscontained a 1:2 ratio of wild-type to heterozygous offspring, but nohomozygous mutants. Of the offspring analyzed by Southern blotting, 29contained only the wild-type allele and 58 were heterozygous for themTim mutation. These results are consistent with mTim being essentialfor mouse survival.

[0233] Heterozygous mTim mutant embryos had reduced mTIM protein levels,confirming the targeting event; wild-type levels=8.04±2.07 (mean±SEM;n=4) versus heterozygote levels=2.98±0.61 (n=5; p<0.05, unpaired ttest). Heterozygous mTim mutants had no obvious developmental orbehavioral abnormalities.

[0234] Heterozygous mTim mutant animals displayed circadian rhythms inlocomotor activity indistinguishable from wild-type mice of isogenicbackground. Rhythmic wheel-running activity of both groups persisted inconstant conditions (>25 days). Furthermore, the period of locomotoractivity was unchanged; wild-type mice displayed a period of 23.52±0.22hrs (n=4) vs. 23.73±0.13 hrs (n=8) for heterozygotes (p>0.05, Student'st-test). The lack of period change in heterozygotes does not rule out aclock-relevant function for mTim, because the null Tim mutation inDrosophila is recessive.

[0235] The mortality rate of homozygous mTim mutant embryos at differentgestational ages was next determined. Histological analysis of embryosfrom 13 litters from heterozygous mTim mutant crosses spanning embryonicday (ED) 6.5 to 11.5 showed a mortality rate of 41%. When corrected fornaturally occurring prenatal attrition (14%, determined fromheterozygous female X wild-type male matings), the lethality rate was25.5%, consistent with the predicted Mendelian rate for a mutation thatis lethal when homozygous.

[0236] Developmental defects due to the mTim mutation were striking atED 7.5. At this stage, presumptive homozygous embryos lack any cellularorganization, with necrotic cell debris filling the amniotic cavity, andresorption by surrounding maternal tissues has already begun.Developmental abnormalities were observed in embryos as early as ED 5.5(data not shown), indicating that mTim is essential for developmentaround the time of implantation. The mechanism behind the essential roleof mTIM for mouse development is currently not known. At ED 7.5, in situhybridization showed that mTim RNA is expressed throughout the embryo,particularly in the embryonic germ cell layers and in the ectoplacentalcone.

[0237] The results show that mTim is essential for embryonicdevelopment.

What is claimed is:
 1. A method for identifying a compound which bindsto a mammalian CRY protein, the method comprising: contacting the CRYprotein with a test compound; and determining whether the CRY proteinbinds to the test compound, wherein binding by the test compound to theCRY protein indicates that the test compound is a CRY protein bindingcompound.
 2. The method of claim 1, wherein the CRY protein is CRY1 orCRY2.
 3. The method of claim 1, wherein the test compound isradiolabeled.
 4. The method of claim 1, further comprising: contactingthe test compound with the CRY protein in the presence of a PER protein;and determining whether the test compound disrupts the association ofthe CRY protein with the PER protein, wherein a decrease in theassociation in the presence of the test compound compared to theassociation in the absence of the test compound indicates that the testcompound disrupts the association of the CRY protein and with PERprotein.
 5. The method of claim 4, wherein the CRY protein is a mouseCRY 1 or CRY2.
 6. The method of claim 4, wherein the PER is a mousePER1, PER2 or PER3.
 7. The method of claim 1, further comprising:contacting the test compound with the CRY protein in the presence of aTIM protein; and determining whether the test compound disrupts theassociation of the CRY protein with the TIM protein, wherein a decreasein the association in the presence of the test compound compared to theassociation in the absence of the test compound indicates that the testcompound disrupts the association of the CRY protein with the TIMprotein.
 8. The method of claim 1, further comprising: contacting thetest compound with the CRY protein in the presence of a CLOCK:BMAL-1complex; and determining whether the test compound disrupts theassociation of the CRY protein with the CLOCK:BMAL-1 complex, wherein adecrease in the association in the presence of the test compoundcompared to the association in the absence of the test compoundindicates that the test compound disrupts the association of the CRYprotein with the CLOCK:BMAL-1 complex.
 9. The method of claim 1, furthercomprising: contacting the test compound with the CRY protein in thepresence of a BMAL-1 protein; and determining whether the test compounddisrupts the association of the CRY protein with the BMAL-1 protein,wherein a decrease in the association in the presence of the testcompound compared to the association in the absence of the test compoundindicates that the test compound disrupts the association of the CRYprotein with the BMAL-1 protein.
 10. The method of claim 1, furthercomprising: contacting the test compound with the first CRY protein inthe presence of a second CRY protein; and determining whether the testcompound disrupts the association of the first CRY protein with thesecond CRY protein, wherein the second CRY protein has an amino acidsequence the same as or different than the first CRY protein, andwherein a decrease in the association in the presence of the testcompound compared to the association in the absence of the test compoundindicates that the test compound disrupts the association of the firstCRY protein and the second CRY protein.
 11. The method of claim 10,wherein the first CRY protein is CRY1 or CRY2.
 12. The method of claim10, wherein the second CRY protein is CRY1 or CRY2.
 13. The method ofclaim 1, further comprising: providing a cell comprising a CRY protein,a CLOCK:BMAL-1 complex, and a DNA comprising an E-box operatively linkedto a reporter gene; introducing the test compound into the cell; andassaying for transcription of the reporter gene in the cell, wherein anincrease in transcription in the presence of the compound compared totranscription in the absence of the compound indicates that the compoundblocks CRY-induced inhibition of CLOCK:BMAL-1-mediated transcription ina cell.
 14. The method of claim 13, wherein the cell is a NIH3T3 cell ora clock neuron.
 15. The method of claim 13, wherein the reporter geneencodes luciferase.
 16. A method for identifying a compound whichdisrupts the association of a CRY protein and a PER protein, the methodcomprising: contacting a test compound with the CRY protein in thepresence of the PER protein; and determining whether the test compounddisrupts the association of the CRY protein and the PER protein, whereina decrease in the association in the presence of the test compoundcompared to the association in the absence of the test compoundindicates that the test compound disrupts the association of the CRYprotein and the PER protein.
 17. The method of claim 16, wherein the CRYprotein is a mouse CRY1 or CRY2.
 18. The method of claim 16, wherein thePER protein is a mouse PER1, PER2 or PER3.
 19. A method for identifyinga compound which disrupts the association of a CRY protein and a TIMprotein, the method comprising: contacting a test compound with the CRYprotein in the presence of the TIM protein; and determining whether thetest compound disrupts the association of the CRY protein and the TIMprotein, wherein a decrease in the association in the presence of thetest compound compared to the association in the absence of the testcompound indicates that the test compound disrupts the association ofthe CRY protein and the TIM protein.
 20. The method of claim 19, whereinthe CRY protein is a mouse CRY1 or CRY2.
 21. The method of claim 19,wherein the TIM protein is a mouse TIM.
 22. A method of identifying acompound that disrupts the association between a CRY protein and aCLOCK:BMAL-1 complex, the method comprising: contacting a test compoundwith the CRY protein in the presence of a CLOCK protein amd a BMAL-1protein; and determining whether the test compound disrupts theassociation of the CRY protein with a complex of the CLOCK protein andthe BMAL-1 protein, wherein a decrease in the association in thepresence of the test compound compared to the association in the absenceof the test compound indicates that the test compound disrupts theassociation of the CRY protein and the CLOCK:BMAL-1 complex.
 23. Themethod of claim 22, wherein the CRY protein is mouse CRY1 or CRY2. 24.The method of claim 22, wherein the CLOCK protein is mouse CLOCK and theBMAL-1 protein is mouse BMAL-1.
 25. A method for identifying a compoundwhich disrupts the association of a CRY protein and a BMAL-1 protein,the method comprising: contacting a test compound with the CRY proteinin the presence of the BMAL-1 protein; and determining whether the testcompound disrupts the association of the CRY protein and the BMAL-1protein, wherein a decrease in the association in the presence of thetest compound compared to the association in the absence of the testcompound indicates that the test compound disrupts the association ofthe CRY protein and the BMAL-1 protein.
 26. The method of claim 25,wherein the CRY protein is a mouse CRY1 or CRY2.
 27. The method of claim25, wherein the BMAL-1 protein is a mouse BMAL-1.
 28. A method foridentifying a compound which disrupts the association of a first CRYprotein and a second CRY protein, the method comprising: contacting atest compound with the first and second CRY proteins; and determiningwhether the test compound disrupts the association of the first CRYprotein with the second CRY protein, wherein the second CRY protein hasan amino acid sequence the same as or different than the first CRYprotein, and wherein a decrease in the association in the presence ofthe test compound compared to the association in the absence of the testcompound indicates that the test compound disrupts the association ofthe first CRY protein with the second CRY protein.
 29. The method ofclaim 28, wherein the first CRY protein is a mouse CRY1 or CRY2.
 30. Themethod of claim 28, wherein the second CRY protein is a mouse CRY1 orCRY2.
 31. A method for identifying a compound that blocks CRYinduced-inhibition of CLOCK:BMAL-1 transcription in a cell, the methodcomprising: providing a cell comprising a CRY protein, a CLOCK:BMAL-1complex, and a DNA comprising an E-box operatively linked to a reportergene; introducing the compound into the cell; and assaying fortranscription of the reporter gene in the cell, wherein an increase intranscription in the presence of the compound compared to transcriptionin the absence of the compound indicates that the compound blocksCRY-induced inhibition of CLOCK:BMAL-1-mediated transcription in a cell.32. The method of claim 31, wherein the cell is a NIH3T3 cell or a clockneuron.
 33. The method of claim 31, wherein the reporter gene encodes aluciferase.
 34. An isolated nucleic acid which encodes a mouse Timprotein.
 35. The nucleic acid of claim 34, wherein the nucleic acidencodes an amino acid sequence which has at least 70% sequence identityto SEQ ID NO:2.
 36. The nucleic acid of claim 34, wherein the nucleicacid encodes the amino acid sequence of SEQ ID NO:2.
 37. A vectorcomprising the nucleic acid of claim
 34. 38. A cell comprising thenucleic acid of claim
 34. 39. A substantially pure preparation of amouse TIM.
 40. A substantially pure antibody which specifically binds tomouse CRY.
 41. A substantially pure antibody which specifically binds tomouse PER.
 42. A substantially pure antibody raised against mouse TIMand which specifically binds to mouse TIM.
 43. A purified preparation ofa mouse CRY:PER heterodimer.
 44. A purified preparation of a CRY:TIMheterodimer.
 45. A purified preparation of a mammalian CRY:CRYhomodimer.
 46. A method for identifying a compound that inhibits thetranscription of Period-2, the method comprising: providing a cellcomprising a Period-2 regulatory sequence operatively linked to areporter gene; introducing a test compound into the cell; and assayingfor transcription of the reporter gene in the cell, wherein a decreasein transcription in the presence of the compound compared totranscription in the absence of the compound indicates that the compoundinhibits Period-2 transcription in a cell.
 47. The method of claim 46,wherein the cell is a NIH3T3 cell, a Cos-7 cell or a clock neuron. 48.The method of claim 46, wherein the reporter gene encodes a luciferase,a chloramphenicol acetyl transferase, a beta-galactosidase, an alkalinephosphate, or a fluorescent protein.
 49. A method for identifying acompound that activates transcription of a Period-2, the methodcomprising: providing a cell comprising a Period-2 regulatory sequenceoperatively linked to a reporter gene; introducing a test compound intothe cell; and assaying for transcription of the reporter gene in thecell, wherein an increase in transcription in the presence of thecompound compared to transcription in the absence of the compoundindicates that the compound activates Period-2 transcription in thecell.
 50. The method of claim 49, wherein the cell is a NIH3T3 cell, aCos-7 cell or a clock neuron.
 51. The method of claim 49, wherein thereporter gene encodes a luciferase, a chloramphenicol acetyltransferase, a beta-galactosidase, an alkaline phosphate, or afluorescent protein.
 52. A method of modulating circadian-clockcontrolled rhythms in a cell comprising introducing into a cell anexpression vector encoding a BMAL-1 protein such that an effectiveamount of the BMAL-1 protein is produced in the cell, thereby modulatingcircadian-clock controlled rhythms.
 53. A method of modulatingcircadian-clock controlled rhythms in a cell comprising introducing intothe cell an effective amount of an oligonucleotide antisense to BMAL-1,thereby inhibiting expression of BMAL-1 in the cell and modulatingcircadian-clock rhythms.
 54. A method of determining if a candidatecompound positively regulates the expression of BMAL-1, the methodcomprising: providing a transgenic animal whose somatic and germ cellscomprise a disrupted Period 2 gene, the disruption being sufficient toinhibit the ability of Period 2 to positively regulate BMAL-1;administering a test compound to the mouse; and detecting BMAL-1expression, wherein an increase in the expression of BMAL-1 indicatesthat the compound positively regulates expression of BMAL-1.